Method for produce sanitation using bacteriophages

ABSTRACT

A method for produce sanitation using bacteriophages is disclosed. According to one embodiment of the present invention, the method includes the steps of (1) providing at least one bacteriophage; and (2) applying the bacteriophage to the produce. The produce may include fruits and vegetables. The produce may be freshly-cut produce, damaged produce, diseased produce, or contaminated produce. The produce may be sprayed with bacteriophage, washed with bacteriphage, immersed in a liquid containing bacteriophage, etc. The bacteriophage may be applied once, periodically or continuously. In one embodiment, chemical sanitizers may also be applied to the produce.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application claims priority from U.S. Provisional PatentApplication No. 60/175,416 filed Jan. 11, 2000, entitled “Method andDevice for Sanitation Using a Bacteriophage” and U.S. Provisional PatentApplication No. 60/205,240 filed May 19, 2000, entitled “Method andDevice for Sanitation Using a Bacteriophage.” The disclosures of theseapplications are incorporated, by reference, in their entireties.

In addition, the present application is related to the following U.S.Provisional Patent Applications: U.S. Provisional Patent Application No.60/175,377 filed Jan. 11, 2000, entitled “Polymer Blends asBiodegradable Matrices for Preparing Biocomposites” and U.S. ProvisionalPatent Application No. 60/175,415 filed Jan. 11, 2000, entitled“Bacteriophage Specific For Vancomycin Resistant Enterococci (VRE).” Thedisclosures of these applications are incorporated, by reference, intheir entireties.

GRANT

This invention was made with Government support under Grant No. ______awarded by the United Statement Department of Agriculture. TheGovernment may have certain rights in the invention.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention is directed the field of bacteriophages.Specifically, it is directed to a method for produce sanitation using abacteriophage.

2. Description of Related Art

Vancomycin-resistant Enterococcus

Over the last ten years there has been an emergence of bacterialpathogens, which demonstrate resistance to many, if not allantimicrobial agents. This is particularly relevant in the institutionalenvironment where nosocomial pathogens are under selective pressure dueto extensive antimicrobial usage. A particular problem in this regardhas been vancomycin-resistant enterococci (VRE), which are not treatablewith standard classes of antibiotics. Despite the recent release of twodrugs to which VRE are susceptible (quinupristin/dalfopristin andlinezolid [Plouffe J F, Emerging therapies for serious gram-positivebacterial infections: A focus on linezolid. Clin Infect dis 2000 Suppl4:S144-9), these microorganisms remain an important cause of morbidityand mortality in immunocompromised patients.

Enterococci are gram positive facultatively anaerobic cocci found in avariety of environmental sources including soil, food and water. Theyare also a common colonizing bacterial species in the human intestinaltract (i.e., the intestinal tract serves as a reservoir for themicroorganism). Although the taxonomy of enterococci has not beenfinalized, it is generally accepted that the genus consists of 19species.

Antibiotic management of serious enterococcal infections has always beendifficult due to the intrinsic resistance of the organisms to mostantimicrobial agents [Arden, R. C, and B. E. Murray, 1994,“Enterococcus: Antimicrobial resistance.” In: Principles and Practice ofInfectious Diseases Update, volume 2, number 4 (February, 1994). NewYork: Churchill Livingstone, Inc. 15 pps; Landman, D., and J. M. Quale,1997, “Management of infections due to resistant enterococci: a reviewof therapeutic options.” J. Antimicrob. Chemother., 40:161-70;Moellering, R. C., 1998, “Vancomcyin-resistant enterococci.” Clin.Infect. Dis. 26:1196-9]. In the 1970's enterococcal infections weretreated with the synergistic combination of a cell wall active agentsuch as penicillin and are aminoglycoside (Moellering, et al. (1971),“Synergy of penicillin and gentamicin against enterococci.” J Infect.Dis., 124:S207-9; Standiford, et al. (1970), “Antibiotic synergism ofenterococci: relation to inhibitory concentrations.” Arch. Intern. Med.,126: 255-9). However, during the 1980's enterococcal strains with highlevels of aminoglycoside resistance and resistance to penicillin,mediated both by a plasmid-encoded β-lactamase and by changes inpenicillin binding proteins, appeared (Mederski-Samoraj, et al. (1983),“High level resistance to gentamicin in clinical isolates ofenterococci.” J. Infect. Dis., 147:751-7; Uttley, et al. (1988),“Vancomycin resistant enterococci.” Lancet i:57-8). In 1988 the firstVRE isolates were identified (Leclercq, et al. (1988), “Plasmid mediatedresistance to vancomycin and teicoplanin in Enterococcus faecium.” NEngl. J: Med., 319:157-61). Such organisms, called VRE because ofresistance to vancomycin, are also resistant to thepenicillin-aminoglyroside combination. VRE includes strains of severaldifferent enterococcal species with clinically significant VREinfections caused by Enterococcus faecium and Enterococcus faecalis.

Enterococci can cause a variety of infections including wound infection,endocarditis, urinary tract infection and bacteremia. AfterStaphylococcus aureus and coagulase negative staphylococci, enterococciare the most common cause of nosocomial bacteremia. Amongimmunocompromised patients, intestinal colonization with VRE frequentlyprecedes, and serves as a risk factor for, subsequent VRE bacteremia(Edmond, et al. (1995), “Vancomycin resistant Enterococcus faeciumbacteremia: Risk factors for infection.” Clin. Inf. Dis., 20:1126-33;Tornieporth, N. G., R. B. Roberts, J. John, A. Hafner, and L. W. Riley,1996, “Risk factors associated with vancomycin-resistant Enterococcusfaecium infection or colonization in 145 matched case patients andcontrol patients.” Clin. Infect. Dis., 23:767-72.]. By using pulse fieldgel electrophoresis as a molecular typing tool investigators at theUniversity of Maryland at Baltimore and the Baltimore VA Medical Centerhave shown VRE strains causing bacteremia in cancer patients are almostalways identical to those which colonize the patients gastrointestinaltract (Roghmann M C, Qaiyumi S, Johnson J A, Schwalbe R, Morris J G(1997), “Recurrent vancomycin-resistant Enterococcus faecium bacteremiain a leukemia patient who was persistently colonized withvancomycin-resistant enterococci for two years.” Clin Infect Dis24:514-5). The risk of acquiring VRE increases significantly when thereis a high rate of VRE colonization among patients on a hospital ward orunit (i.e., when there is high “colonization pressure”). In one study inthe Netherlands, colonization pressure was the most important variableaffecting acquisition of VRE among patients in an intensive care unit(Bonten M J, et al, “The role of “colonization pressure” in the spreadof vancomycin-resistant enterococci: an important infection controlvariable.” Arch Intern Med 1998; 25:1127-32). Use of antibiotics hasbeen clearly shown to increase the density, or level of colonization, inan individual patient (Donskey C J et al, “Effects of antibiotic therapyon the density of vancomycin-resistant enterococci in the stool ofcolonized patients.” N Engl J Med 2000;343:1925-32): this, in turn,would appear to increase the risk of subsequent infection, and the riskof transmission of the organism to other patients.

Multi-Drug Resistant Staphylococcus aureus (MDRSA)

S. aureus is responsible for a variety of diseases ranging from minorskin infections to life-threatening systemic infections, includingendocarditis and sepsis [Lowy, F. D., 1998, “Staphylococcus aureusinfections.” N. Engl. J. Med, 8:520-532]. It is a common cause ofcommunity- and nosocomially-acquired septicemia (e.g., of approximately2 million infections nosocomially acquired annually in the UnitedStates, approximately 260,000 are associated with S. aureus [Emori, T.G., and R. P. Gaynes, 1993, “An overview of nosocomial infections,including the role of the microbiology laboratory,” Clin. Microbiol.Rev., 4:428-442]). Also, approximately 20% of the human population isstably colonized with S. aureus, and up to 50% of the population istransiently colonized, with diabetics, intravenous drug users, patientson dialysis, and patients with AIDS having the highest rates of S.aureus colonization [Tenover, F. C., and R. P. Gaynes, 2000, “Theepidemiology of Staphylococcus infections,” p. 414-421, In: V. A.Fischetti, R. P. Novick, J. J. Ferretti, D. A. Portnoy, and J. I. Rood(ed), Gram-positive pathogens, American Society for Microbiology,Washington, D.C.]. The organism is responsible for approximatelyone-half of all skin and connective tissue infections, includingfolliculitis, cellulitis, furuncules, and pyomyositis, and is one of themost common causes of surgical site infections. The mortality rate forS. aureus septicemia ranges from 11 to 48% [Mortara, L. A., and A. S.Bayer, 1993, “Staphylococcus aureus bacteremia and endocarditis. Newdiagnostic and therapeutic concepts.” Infect. Dis. Clin. North. Am.,1:53-68].

Methicillin was one of the first synthetic antibiotics developed totreat penicillin-resistant staphylococcal infections. However, theprevalence of methicillin-resistant S. aureus strains or “MRSA” (whichalso are resistant to oxacillin and nafcillin) has drastically increasedin the United States and abroad [Panlilio, A. L., D. H. Culver, R. P.Gaynes, S. Banerjee, T. S. Henderson, J. S. Tolson, and W. J. Martone,1992, “Methicillin-resistant Staphylococcus aureus in U.S. hospitals,1975-1991.” Infect. Control Hosp. Epidemiol., 10:582-586]. For example,according to the National Nosocomial Infections Surveillance System[National Nosocomial Infections Surveillance (NNIS) report, data summaryfrom October 1986-April 1996, issued May 1996, “A report from theNational Nosocomial Infections Surveillance (NNIS) System.” Am. J.Infect. Control., 5:380-388], approximately 29% of 50,574 S. aureusnosocomial infections from 1987 to 1997 were resistant to the β-lactamantibiotics (e.g., oxacillin, nafcillin, methicillin), and the percentof MRSA strains among U.S. hospitals reached approximately 40% by theend of the same period. At the University of Maryland MedicalCenter, >50% of all S. aureus blood isolates are now methicillinresistant.

In this setting, there is great concern about the possible emerge ofmethicillin-resistant/multi-drug resistant S. aureus strains which arevancomycin resistant—and which would be essentially untreatable.Although overt resistance to vancomycin has not yet been documented inclinical isolates, there have been several reports of clinicalinfections with S. aureus strains having intermediate resistance tovancomycin (MICs=8 μg/ml), which suggests that untreatablestaphylococcal infections may not be too far away [Tenover, F. C., andR. P. Gaynes. 2000]. Given the virulence of S. aureus, the emergence ofsuch untreatable strains would be devastating and have a major impact onthe way in which medicine is practiced in this country.

Staphylococcal species, including MDRSA, are common colonizers of thehuman nose; in one community-based study, 35% of children and 28% oftheir, guardians had nasal Staphylococcus aureus colonization (ShopsinB, et al, “Prevalence of methicillin-resistant andmethicillin-susceptible Staphylococcus aureus in the community.” JInfect Dis 2000;182:359-62.). Persons who are nasally colonized withMRSA have an increased risk of developing serious systemic infectionswith this microorganism, and, in particular, colonization or priorinfection with MDRSA significantly increases the risk of subsequentbacteremia with MDRSA (Roghmann M C, “Predicting methicillin resistanceand the effect of inadequate empiric therapy on survival in patientswith Staphylococcus aureus bacteremia. Arch Intern Med 2000;160:1001-4).As seen with VRE, the rate of colonization of persons with MDRSA on aunit (the colonization pressure) significantly increases the risk ofacquisition of MDRSA for other patients on the unit (Merrer J, et al,“Colonization pressure” and risk of acquisition of methicillin-resistantStaphylococcus aureus in a medical intensive care unit.” Infect ControlHosp Epidemiol 2000;21:718-23).

Multi-drug Resistant Pseudomonas aeruginosa

Pseudomonas aeruginosa is a highly virulent gram-negative bacterialspecies that is responsible for bacteremia, wound infections, pneumonia,and urinary tract infections. Increasing problems with multi-antibioticresistance in Pseudomonas has been noted in hospitals, with particularconcern focusing on strains which are generally designated as“Imipenem-resistant Pseudomonas”, reflecting the last majorantimicrobial agent to which they have become resistant. Many of thesestrains are resistant to all major antibiotic classes, presentingsubstantive difficulties in management of infected patients.

As seen with other Gram-negative microorganisms, Pseudomonas strainsoften emerge as the primary colonizing flora of the posterior pharynxduring hospitalization. Strains present in the posterior pharynx, inturn, are more likely to be aspirated into the lungs, and causepneumonia. In this setting, colonization with multi-drug resistantPseudomonas represents a potentially serious risk factor for developmentof multi-drug resistant Pseudomonas pneumonia.

Bacteriophage

Bacteriophage has been used therapeutically for much of this century.Bacteriophage, which derive their name from the Greek word “phago”meaning “to eat” or “bacteria eaters”, were independently discovered byTwort and independently by D'Herelle in the first part of the twentiethcentury. Early enthusiasm led to their use as both prophylaxis andtherapy for diseases caused by bacteria. However the results from earlystudies to evaluate bacteriophage as antimicrobial agents were variabledue to the uncontrolled study design and the inability to standardizereagents. Later in well designed and controlled studies it was concludedthat bacteriophage were not useful as antimicrobial agents (Pyle, N. J.(1936), J. Bacteriol., 12:245-61; Colvin, M. G. (1932), J. Infect Dis.,51:17-29; Boyd et al. (1944), Trans R. Soc. Trop. Med. Hyg., 37:243-62).

This initial failure of phage as antibacterial agents may have been dueto the failure to select for phage that demonstrated high in vitro lyticactivity prior to in vivo use. For example, the phage employed may havehad little or no activity against the target pathogen, were used againstbacteria that were resistant due to lysogenization or the phage itselfmight be lysogenic for the target bacterium (Barrow, et al. (1997),“Bacteriophage therapy and prophylaxis: rediscovery and renewedassessment of potential.” Trends in Microbiology, 5:268-71). However,with a better understanding of the phage-bacterium interaction and ofbacterial virulence factors, it was possible to conduct studies whichdemonstrated the in vivo anti-bacterial activity of the bacteriophage(Asheshov, et al. (1937), Lancet, 1:319-20; Ward, W. E. (1943), J.Infect. Dis., 72:172-6; Lowbury, et al. (1953), J. Gen. Microbiol.,9:524-35). In the U.S. during the 1940's Eli Lilly commerciallymanufactured six phage products for human use including preparationstargeted towards staphylococci, streptococci and other respiratorypathogens.

With the advent of antibiotics, the therapeutic use of phage graduallyfell out of favor in the U.S. and Western Europe and little subsequentresearch was conducted. However, in the 1970's and 1980's there werereports of bacteriophage therapy continuing to be utilized in EasternEurope, most notably in Poland and the former Soviet Union.

Phage therapy has been used in the former Soviet Union and EasternEurope for over half a century, with research and production centered atthe Eliava Institute of Bacteriophage in Tbilisi, in what is now theRepublic of Georgia. The international literature contains severalhundred reports on phage therapy, with the majority of the publicationscoming from researchers in the former Soviet Union and eastern Europeancountries. To give but a few examples, phages have been reported to beeffective in treating (i) skin and blood infections caused byPseudomonas, Staphylococcus, Klebsiella, Proteus, and E. coli [Cislo,M., M. Dabrowski, B. Weber-Dabrowska, and A. Woyton, 1987,“Bacteriophage treatment of suppurative skin infections,” 35(2):175-183;Slopek, S., I. Durlakowa, B. Weber-Dabrowska, A. Kucharewicz-Krukowska,M. Dabrowski, and R. Bisikiewicz, 1983, “Results of bacrteriophagetreatment of suppurative bacterial infections. I. General evaluation ofthe results,” Archivum. Immunol. Therapiae Experimental, 31:267-291;Slopek, S., B. Weber-Dabrowska, M. Dabrowski, and A.Kucharewicz-Krukowska, 1987, “Results of bacteriophage treatment ofsuppurative bacterial infections in the years 1981-1986,”, 35:569-83],(ii) staphylococcal lung and pleural infections [Meladze, G. D., M. G.Mebuke, N. S. Chkhetia, N. I. Kiknadze, G. G. Koguashvili, I. I.Timoshuk, N. G. Larionova, and G. K. Vasadze, 1982, “The efficacy ofStaphylococcal bacteriophage in treatment of purulent diseases of lungsand pleura,” Grudnaya Khirurgia, 1:53-56 (in Russian, summary inEnglish)], (iii) P. aeruginosa infections in cystic fibrosis patients[Shabalova, I. A., N. I. Karpanov, V. N. Krylov, T. O. Sharibjanova, andV. Z. Akhverdijan. “Pseudomonas aeruginosa bacteriophage in treatment ofP. aeruginosa infection in cystic fibrosis patients,” abstr. 443. InProceedings of IX international cystic fibrosis congress, Dublin,Ireland], (iv) neonatal sepsis [Pavlenishvili, I., and T. Tsertsvadze.1985. “Bacteriophage therapy and enterosorbtion in treatment of sepsisof newbornes caused by gram-negative bacteria.” In abstracts, p. 104,Prenatal and Neonathal Infections, Toronto, Canada], and (v) surgicalwound infections [Peremitina, L. D., E. A. Berillo, and A. G. Khvoles,1981, “Experience in the therapeutic use of bacteriophage preparationsin supportive surgical infections.” Zh. Mikrobiol. Epidemiol.Immunobiol. 9:109-110 (in Russian)]. Several reviews of the therapeuticuse of phages were published during the 1930s-40s [Eaton, M. D., and S.Bayne-Jones, 1934, “Bacteriophage therapy: review of the principles andresults of the use of bacteriophage in the treatment of infections,” J.Am. Med. Assoc., p. 103; Krueger, A. P., and E. J. Scribner, 1941, “Thebacteriophage: its nature and its therapeutic use,” J. Am. Med. Assoc.,p. 116] and recently [Barrow, P. A., and J. S. Soothill, 1997,“Bacteriophage therapy and propylaxis—rediscovery andf renewedassessment of potential,” Trends in Microbiol., 5(7):268-271; Lederberg,J., 1996, “Smaller fleas . . . ad infinitum: therapeutic bacteriophage,”Proc. Natl. Acad. Sci. USA, 93:3167-3168]. In a recent paper publishedin the Journal of Infection (Alisky, J., K. Iczkowski, A. Rapoport, andN. Troitsky, 1998, “Bacteriophages show promise as antimicrobialagents,” J. Infect., 36:5-15), the authors reviewed Medline citations(published during 1966-1996) of the therapeutic use of phages in humans.There were twenty-seven papers from Britain, the U.S.A., Poland and theSoviet Union, and they found that the overall reported success rate forphage therapy was in the range of 80-95%.

These are several British studies describing controlled trials ofbacteriophage raised against specific pathogens in experimentallyinfected animal models such as mice and guinea pigs (See, e.g., Smith.H. W., and M. B. Huggins “Successful treatment of experimentalEscherichia coli infections in mice using phages: its generalsuperiority over antibiotics” J. Gen. Microbial., 128:307-318 (1982);Smith, H. W., and M. B. Huggins “Effectiveness of phages in treatingexperimental E. coli diarrhea in calves, piglets and lambs” J. Gen.Microbiol., 129:2659-2675 (1983); Smith, H. W. and R. B. Huggins “Thecontrol of experimental E. coli diarrhea in calves by means ofbacteriophage”. J. Gen. Microbial., 133:1111-1126 (1987); Smith, H. W.,R. B. Huggins and K. M. Shaw “Factors influencing the survival andmultiplication of bacteriophages in calves and in their environment” J.Gen. Microbial., 133:1127-1135 (1987)). These trials measured objectivecriteria such as survival rates. Efficacy against Staphylococcus,Pseudomonas and Acinetobacter infections were observed. These studiesare described in more detail below.

One U.S. study concentrated on improving bioavailability of phage inlive animals (Merril, C. R., B. Biswas, R. Carlton, N. C. Jensen, G. J.Greed, S. Zullo, S. Adhya “Long-circulating bacteriophage asantibacterial agents” Proc. Natl. Acad Sci. USA, 93:3188-3192 (1996)).Reports from the U.S. relating to bacteriophage administration fordiagnostic purposes have indicated phage have been safely administeredto humans in order to monitor humoral immune response in adenosinedeaminase deficient patients (Ochs, et al. (1992), “Antibody responsesto bacteriophage phi X174 in patients with adenosine deaminasedeficiency.” Blood, 80:1163-71) and for analyzing the importance of cellassociated molecules in modulating the immune response in humans (Ochs,et al. (1993), “Regulation of antibody responses: the role of complementacrd adhesion molecules.” Clin. Immunol. Immunopathol., 67:S33-40).

Additionally, Polish, Georgian, and Russian papers describe experimentswhere phage was administered systemically, topically or orally to treata wide variety of antimicrobial resistant pathogens (See, e.g.,Shabalova, I. A., N. I. Karpanov, V. N. Krylov, T. O. Sharibjanova, andV. Z. Akhverdijan. “Pseudomonas aeruginosa bacteriophage in treatment ofP. aeruginosa infection in cystic fibrosis patients,” Abstr. 443. InProceedings of IX International Cystic Fibrosis Congress, Dublin,Ireland; Slopek, S., I. Durlakowa, B. Weber-Dabrowska, A.Kucharewicz-Krukowska, M. Dabrowski, and R Bisikiewicz. 1983. “Resultsof bacteriophage treatment of suppurative bacterial infections. I.General evaluation of the results.” Archivum, Immunol. TherapiaeExperimental, 31:267-291; Slopek, S., B. Weber-Dabrowska, M. Dabrowski,and A. Kucharewicz-Krukowska. 1987. “Results of bacteriophage treatmentof suppurative bacterial infections in the years 1981-1986”, ArchivumImmunol. Therapiae Experimental, 35:569-83.

Infections treated with bacteriophage included osteomyelitis, sepsis,empyema, gastroenteritis, suppurative wound infection, pneumonia anddermatitis. Pathogens involved included Staphylococci, Sreptococci,Klebsiella, Shigella, Salmonella, Pseudomonas, Proteus and Escherichia.These articles reported a range of success rates for phage therapybetween 80-95% with only rare reversible allergic or gastrointestinalside effects. These results indicate that bacteriophage may be a usefuladjunct in the fight against bacterial diseases. However, thisliterature does not describe, in any way anticipate, or otherwisesuggest the use of bacteriophage to modify the composition of colonizingbacterial flora in humans, thereby reducing the risk of subsequentdevelopment of active infections.

Salmonella in Humans

Salmonella are the leading cause of food-borne disease in the UnitedStates. In 1993, USDA estimated that there were between 700,000 and 3.8million Salmonella cases in this country, with associated medical costsand productivity losses of between $600 million and $3.5 billion. SeeFood Safety and Inspection Service, 1995; 9 CFR Part 308; PathogenReduction; Hazard Analysis and Critical Control Point (HACCP) Systems;Proposed Rule 60 Fed. Reg. 6774-6889; FoodNet, unpublished data. Moreexact estimates of incidence have come from CDC's FoodNet system, basedon active surveillance data from seven sentinel sites, with the mostrecent data suggesting that there are 1.4 million cases annually. SeeMead, P. S., L. Slutsker, V. Dietz, L. F. McCaig, J. S. Bresee, C.Shapiro, P. M. Griffin, and R. V. Tauxe “Food-related illness and deathin the United States” Emerg. Infec. Dis. 5:607-625 (1999). While allSalmonella appear to be able to cause illness, S. typhimurium and S.enteritidis accounted for 22.6% and 22% of all human cases,respectively, in the United States between 1991 and 1995. See Centersfor Disease Control and Prevention “Salmonella Surveillance, AnnualSummary” 1991, 1992, 1993-1995.

S. typhimurium has become of particular concern because of the recentemergence of a highly antibiotic resistant strain (resistant toampicillin, chloramphenicol, streptomycin, sulfonamides, andtetracycline) designated as definitive type 104 (DT104). In 1979-80,this resistance pattern was seen in 0.6% of S. typhimurium isolates; by1996, 34% of all U.S. isolates tested by public health laboratories hadthis pattern, with further testing showing that approximately 90% ofthese resistant isolates were DT104. See Glynn, M. K., C. Bopp, W.DeWitt, P. Dabney, M. Mokhtar, and F. J. Angulo “Emergence ofmultidrug-resistant Salmonella enterica serotype typhimurium DT104infections in the United States” N. Eng. J. Med. 19:1333-8 (1988).Recent data also suggest that DT-104 is beginning to acquire resistanceto trimethoprim and quinolones. See Wall, P. G., D. Morgan, K. Lamden.M. Ryan, M. Griffin, E. J. Threlfall, L. R. Ward, and B. Rowe “A casecontrol study of infection with an epidemic strain of multiresistantSalmonella typhimurium DT104 in England and Wales” Commun. Dis. Rep. CDRRev. 4:R130-8135 (1994). While data on pathogenicity are limited, DT104appears to be responsible for increased human morbidity and mortality,as compared with other Salmonella. See Centers for Disease Control“Multidrug resistant Salmonella serotype typhimurium—United States,1996” Morbid Mortal Weekly Rep. 46:308-10 (1997).

Among S. enteritidis isolates, attention has focused on phage types 8and 4. Phage type 8 accounts for approximately half of all U.S. S.enteritidis isolates. See Hickman-Brenner, F. W., A. D. Stubbs, and J.J. Farmer, III “Phage typing of Salmonella enteritidis in the UnitedStates” J. Clin. Microbiol., 29;2817-23 (1991); Morris, J. G., Jr., D.M. Dwyer, C. W. Hoge, A. D. Stubbs, D. Tilghman, C. Groves, E. Israel,and J. P. Libonati “Changing clonal patterns of Salmonella enteritidisin Maryland: An evaluation of strains isolated between 1985-90” J. Clin.Microbiol., 30:1301-1303 (1992). Phage type 4 is seen less frequently,but has been associated with recent major outbreaks; it clearly hasincreased virulence in chickens, and, again, may have increasedvirulence in humans. See Humphrey T. J., Williams A., McAlpine K., LeverM. S., Guard-Petter J., and J. M. Cox “Isolates of Salmonella entericaEnteritidis PT4 with enhanced heat and acid tolerance are more virulentin mice and more invasive in chickens” Epidemiol. Infect. 117:79-88(1996); Rampling, A., J. R. Anderson, R. Upson, E. Peters, L. R. Ward,and B. Rowe “Salmonella enteritidis phage type 4 infection of broilerchickens: a hazard to public health” Lancet, ii:436-8 (1989).

In healthy adults, Salmonella generally causes a self-limited diarrhealillness; however, these individuals may asymptomatically carry theorganism in their intestinal tract for six months or more aftercessation of symptoms (convalescent carriage), serving as one source forcontinue transmission of the organism in the community. The elderly, thevery young, and persons who are immunocompromised are at risk forSalmonella bacteremia, which may occur in as many as 5% of infected“high risk” patients. See Taylor, J. L., D. M. Dwyer, C. Groves, A.Bailowitz, D. Tilghman, V. Kim, A. Joseph, and J. G. Morris, Jr.“Simultaneous outbreak of Salmonella enteritidis and Salmonellaschwarzengrund in a nursing home: association of S. enteritidis withbacteremia and hospitalization” J. Infect. Dis. 167:781-2 (1993).Between 1% and 3% of infected persons may also develop reactivearthritis, with the possibility of associated long-term disability.

Antibiotic therapy of diarrheal illness is not effective, and mayactually prolong intestinal carriage. See Alavidze, Z., and I. Okolov“Use of specific bacteriophages in prophylaxis of intrahospitalinfections caused by P. aeruginosa” In: Abst., All-Soviet Unionconference “Modern biology at the service of public health,” Kiev,Ukraine (1988). Bacteremia is, obviously, treated with antibiotics,although the emergence of highly resistant strains such as DT104 hasbegun to create problems in patient management. See Wail, P. G., D.Morgan, K. Lamden, M. Ryan, M. Griffin, E. J. Threlfall, L. R. Ward, andB. Rowe “A case control study of infection with an epidemic strain ofmultiresistant Salmonella typhimurium DT104 in England and Wales”Commun. Dis. Rep. CDR Rev. 4-R130-RI35 (1994). There is currently noeffective means of limiting or eradicating carriage of the organism inthe intestinal tract. See Neill, M. A., S. M. Opal, J. Heelan, R.Giusti, J. E. Cassidy, R. White, and K. H. Mayer “Failure ofciprofloxacin to eradicate convalescent fecal excretion after acuteSalmonellosis: experience during an outbreak in health care workers”Ann. Intern. Med. 119:195-9 (1991).

Salmonella in Chickens

USDA estimates that in 50-75% of human Salmonella cases themicroorganism is acquired from meat, poultry, or eggs, with poultryserving as the primary vehicle of transmission. Salmonella are part ofthe normal, colonizing intestinal flora in many animals, includingchickens. Studies conducted in the early 1990's by USDA indicated that20-25% of broiler carcasses and 18% of turkey carcasses werecontaminated with Salmonella prior to sale. See Food Safety andInspection Service (1995); 9 CFR Part 308; Pathogen Reduction; HazardAnalysis and Critical Control Point (HACCP) Systems; Proposed Rule; 60Fed. Reg. 6774-6889.

Contamination may result from rupture of the intestinal tract duringslaughter. However, with current slaughter techniques, removal of theviscera seldom results in intestinal rupture and carcasscontamination—and, when it does occur, the carcass is immediately taggedfor “reprocessing.” The more common source of Salmonella is the skin ofthe animal itself, with the feather follicles serving as a sanctuary forbacteria. In contrast to beef, chickens are slaughtered “skin on,” sothat antemortem contamination of feathers becomes an important elementin determining whether Salmonella can be isolated from the carcass. Theclose quarters in chicken houses, and the piling of chicken crates ontrucks on the way to slaughterhouses, results in frequent contaminationof feathers by feces. If members of a flock have high levels ofintestinal colonization with Salmonella, there are multipleopportunities for contamination of feathers and feather follicles withthe microorganism, and, in turn, for Salmonella contamination of thefinal product.

According to the CDC FoodNet/Salmonella surveillance system, the fivemost common human Salmonella isolates in the United States during1990-1995 were S. typhimurium, S. enteritidis, S. heidelberg, S.newport, and S. hadar. Further, according to the USDA/FSIS data, thefive most common Salmonella serotypes isolated from broiler chickensduring the same period were S. heidelberg, S. kentucki, S. hadar, S.typhimurium, and S. thomson. While Applicants do not consider this to bean exhaustive list, Applicants note that these are common Salmonellaisolates and serotypes.

The rate of Salmonella contamination of poultry carcasses was a majorfocus of the recently implemented revision of the national food safetyregulations (Pathogen Reduction; Hazard Analysis and Critical ControlPoint (HACCP) Systems), which mandates government testing for Salmonellain all slaughter plants. Regulations now in effect require that productbe tested by putting a whole chicken carcass in a “baggie” with culturemedia and shaking; growth of any Salmonella from broth counts as apositive test. Plants must meet specific standards for percentage ofproduct contaminated, based on national averages; failure to meet thesestandards results in plant closure. See Food Safety Inspection Service(1996); 9 CFR Part 304, et seq.; Pathogen Reduction; Hazard Analysis andCritical Control Point (HACCP) Systems; Final Rule 61 Fed. Reg.38806-989. Concerns about Salmonella contamination have also become amajor issue in international trade, with Russia and other countrieshaving embargoed millions of dollars worth lots of chickens because ofidentification of Salmonella in the product.

In this environment, there are strong public health, regulatory, andtrade incentives for producers to reduce levels of Salmonellacontamination in poultry. Irradiation of raw product (i.e., chickencarcasses) is efficacious, but expensive, and is limited by the smallnumber of irradiation facilities and by consumer acceptance. Treatmentof chickens with antibiotics does not eradicate colonization, tendingsimply to select out for more resistant organisms. Antibiotics (incontrast to phage) generally have activity against multiple bacterialspecies; their administration can result in serious perturbations in themicrobial ecology of the animal's intestinal tract, with accompanyingloss of “colonization resistance” and overgrowth of microorganisms thatare resistant to the antimicrobial agent used. Vaccination is similarlyineffective in elimination of Salmonella. See Hassan, J. O., and R.Curtiss, III “Efficacy of a live avriulent Salmonella typhimuriumvaccine in preventing colonization and invasion of laying hens bySalmonella typhimurium and Salmonella enteritidis” Avian. Dis. 41:783-91(1997); Methner, U., P. A. Barrow, G. Martin, and H. Meyer “Comparativestudy of the protective effect against Salmonella colonization in newlyhatched SPF chickens using live, attenuated Salmonella vaccine strains,wild-type Salmonella strains or a competitive exclusion product” Int. J.Food Microbiol., 35:223-230 (1997); Tan, S., C. L. Gyles, and B. N.Wilkie “Evaluation of an aroA mutant Salmonella typhimurium vaccine inchickens using modified semisolid Rappaport Vassiliadis medium tomonitor fecal shedding” Vet. Microbiol., 54:247-54 (1997).

Competitive exclusion (i.e., administration of “good” bacteria to “crowdout” Salmonella and other “bad” bacteria) has shown variable success.See Palmu, L, I. Camelin “The use of competative exclusion in broilersto reduce the level of Salmonella contamination on the farm and at theprocessing plant” Poultry Sci. 76:1501-5 (1997). There is now acommercially available competitive exclusion product, PreEmpt (producedby MS Bioscience), that consists of 27 different bacteria strains- Inpreliminary testing, it appears to be effective in limiting Salmonellacolonization, but its usage is hampered by the cost. Most importantly,its efficacy is significantly decreased if antibiotics are administeredto animals as growth additives (a standard practice in the poultryindustry).

In the absence of any other definitive means of eradicating theorganism, USDA has articulated the concept of Salmonella control througha “multiple hurdle” approach, encouraging implementation of proceduresto reduce the risk of contamination during slaughter while at the sametime seeking to limit colonization/contamination of broiler flocks bythe organism. Under these circumstances, there is a clear market forproducts and approaches that can be used as part of an overall programof Salmonella control. Any such product should be cheap, safe, and easyto use-, there would also be potential advantages for products whichcould be targeted toward specific pathogens, such as S. enteritidis PT4and S. typhimurium DT104.

SUMMARY OF THE INVENTION

Therefore, a need has arisen for a method for produce sanitation usingbacteriophage.

According to one embodiment of the present invention, a method forsanitation of produce using at least one bacteriophage is disclosed. Themethod includes the steps of (1) providing at least one bacteriophage;and (2) applying the bacteriophage to the produce. The produce mayinclude fruits and vegetables.

The produce may be freshly-cut produce, damaged produce, diseasedproduce, or contaminated produce. The produce may be sprayed withbacteriophage, washed with bacteriphage, immersed in a liquid containingbacteriophage, etc. The bacteriophage may be applied once, periodicallyor continuously.

In one embodiment, chemical sanitizers may also be applied to theproduce.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic of a poultry processing scheme according to oneembodiment of the present invention.

DETAILED DESCRIPTION OF THE EMBODIMENTS

Bacteriophage technology can be of value in managing a large variety ofbacterial infections because: (i) bacteriophages are highly specific andvery effective in lysing targeted pathogenic bacteria, (ii)bactenrophages are absolutely specific for prokaryotes, and do notaffect humans or animals, (iii) bacteriophages are safe, as underscoredby their extensive clinical use in Eastern Europe and the former SovietUnion, and the commercial sale of phages in the 1940's in the UnitedStates, (iv) phage preparations can rapidly be modified to combat theemergence of newly arising bacterial threats, and (v) phage productionis seen to be cost-effective for large-scale applications in a varietyof medical settings. Of particular relevance, bacteriophage will notkill non-pathogenic, “normal flora” bacteria, thereby retaining the“colonization resistance” of reservoirs such as the human intestinaltract, the nose, and the posterior pharynx. Accordingly, the presentinvention envisions using lytic phages (in combination with antibioticsor alone) to prophylactically or therapeutically eliminate variousbacteria capable of causing diseases of the gastrointestinal,genitourinary, and respiratory tracts, and skin, oral cavity, andbloodstream. In accordance with this invention, therapeutic phages canbe administered in a number of ways, in various formulations, including:(i) orally, in tablets or liquids, (ii) locally, in tampons, rinses orcreams, (iii) aerosols, and (iv) intravenously.

One benefit of bacteriophage therapy when compared to antibiotic therapyrelates to the relative specificity of the two therapeutic modalities.Bacteriophage are specific for particular bacterial strains or species,while antibiotics typically are broadly effective against a largemultiplicity of bacterial species or genera. It is well known thatnormal individuals are colonized with innocuous bacteria, and thiscolonization may be beneficial to the colonized individual (see U.S.Pat. No. 6,132,710, incorporated herein by reference). Antibiotictherapy can severely alter colonization or even eliminate beneficialcolonization completely. This often has have adverse effects, such asthe outgrowth of opportunistic species such as Clostridium difficile,which then leads to an antibiotic-associated colitis. Similarly,antibiotic therapy with its well-known adverse effect upon colonizationwith normal flora leads to increased density of VRE colonization (seeDonskey V. J. et al., Effect of Antibiotic Therapy on the Density ofVancomycin-Resistant Enterococci in the Stool of Colonized Patients. NewEngland Journal of Medicine, 2000, 343:1925-1932.) In contrast,bacteriophage therapy specifically affects the bacterial strains thatare sensitive or susceptible to lytic infection by the particularbacteriophage in the therapeutic composition, but leaves other(innocuous or beneficial) bacteria unaffected. Thus, bacteriophagetherapy is preferable for prophylactic treatment where alteration ofnormal microflora should be minimized.

In a preferred mode of this invention, phage technology is focused ontwo important human pathogens, VRE and MDRSA, and the value of VRE- andMDRSA-specific lytic phages in different settings: (i) oraladministration of phages for prophylaxis against septicemia, (ii) localapplication of phages for prophylaxis/treatment of skin and woundinfections, (iii) intravenous administration of phages for therapy ofsepticemia, and (iv) the use of aerosolized phages against respiratorypathogens.

VRE infection has become a particularly serious problem amongimmunocompromised and/or seriously ill patients in intensive care units,cancer centers and organ transplant units. Since VRE are resistant toall currently used antimicrobials, alternate approaches to reducing oreliminating VRE gastrointestinal colonization in immunocompromisedpatients must be found in order to reduce the prevalence of VREbacteremia. Oral administration of lytic bacteriophage active againstVRE is one such approach.

The general rule is that patients first become colonized by pathogenicbacteria present in their immediate environment before developingillness due to those bacteria. Serious VRE infections, includingsepticemia, usually are preceded by intestinal colonization with theinfecting organisms; therefore, the risk of septicemia is likely to bedecreased by reducing colonization prior to periods when patients areseverely neutropenic or otherwise immunosuppressed (i.e., reducingintestinal colonization may also reduce the risk of bloodstreaminvasion). The present inventors have discovered that certain strains ofbacteriophage are particularly effective at lysing VRE. By administeringthese VRE-active bacteriophage to persons colonized with VRE, it ispossible to substantially reduce or even eliminate VRE from thecolonized person. Thus, the present invention provides strains of phagewhich are particularly effective against VRE, methods for obtainingadditional strains of VRE-active phage, methods for treating patientscolonized with VRE by administering VRE-active phage, and methods ofreducing nosicomial infection rate by administering VRE-active phage invivo, ex vivo, or both, to selected locations, areas, objects and/orpersons.

Analogous approaches using bacteriophage targeted to other pathogenicbacteria are also contemplated by this invention. S. aureus phagepreparations can reduce contamination of skin and wounds with S. aureus,which in turn may prevent the development of serious surgical siteinfections and septicemia. Phage active against Pseudomonas species canbe used to reduce colonization that threatens to develop into pneumoniain immunocompromised patients or in individuals suffering from cysticfibrosis.

VRE-active Bacteriophage

The present inventors have isolated several lytic phages active againstgenetically diverse (as assessed by pulsed field gel electrophoresisand/or arbitrary pruned polymerase chain reaction or other nucleic acidamplification techniques) VRE strains. In vitro susceptibility testsinvolving 234 VRE strains (184 E. faecium, 41 E. faecalis and 6 E.gallinarium isolated from patients at the University of Maryland and theBaltimore VA Medical Center, and 3 E. faecium ATCC strains), resulted inthe Intralytix phage collection being able to cumulatively lyse all VREstrains in the collection, with one particular phage being able to lyse95% of VRE strains. Furthermore mice whose gastrointestinal tract wascolonized with VRE under selective pressure of antibioticadministration, were orogastrically administered VRE-active phages,which resulted in a 1 to 3 log reduction of VRE gastrointestinalcolonization compared to a control group of animals not given phage.This occurred within a 48 to 72 hour time frame. No side effects due tothe phage were observed.

Bacteriophage strains may be isolated by analogous procedures to thoseused to isolate the VRE-active strains described herein. Suitablebacteriophage may be isolated from any sample containing bacteriophage,which typically are found in association with their host bacteria. Thus,any source that might be expected to contain VRE is suitable for use asa source of VRE-active bacteriophage. Such samples include fecal, urine,or sputum samples from patients, particularly patients undergoing acuteor prophylactic antibiotic therapy, patients in intensive care units orimmunocompromised patients. Such patients may include but are notlimited to burn patients, trauma patients, patients receiving bonemarrow and/or organ transplants, cancer patients, patients withcongenital or acquired immunodeficiency diseases, dialysis patients,liver disease patients, and patients with acute or chronic renalfailure. Body fluids including ascites, pleural effusions, jointeffusions, abscess fluids, and material obtained from wounds. Whilehumans are the primary reservoir for VRE, the organism also can bereadily found in the immediate environment of infected/colonizedpatients such as bedrails, bed sheets, furniture, etc. (Bodnar, U. R. etal (1996), “Use of in house studies of molecular epidemiology and fullspecies identification of controlling spread of vancomycin resistantEnterococcus faecalis isolates”, J. Clin. Microbiol., 34: 2129-32;Bonten, M. J. M. et al (1996), “Epidemiology of colonization of patientsand the environment with vancomycin resistant enterococci.” Lancet, 348:1615-19; Noskin, G. A. (1995), “Recovery of vancomycin resistantenterococci on fingertips and environmental surfaces.” Infect. ControlHosp. Epidemiol., 16: 577-81). Consequently, samples for bacteriophageisolation may also be obtained from nonpatient sources, includingsewage, especially sewage streams near intensive care units or otherhospital venues, or by swab in hospital areas associated with risk ofnosicomial infection, such as intensive care units. Other suitablesampling sites include nursing homes, rest homes, military barracks,dormitories, classrooms, and medical waste facilities. Phages also canbe isolated from rivers and lakes, wells, water tables, as well as otherwater sources (including salt water). Preferred sampling sites includewater sources near likely sites of contamination listed above.

Suitable methods for isolating pure bacteriophage strains from abacteriophage-containing sample are well known, and such methods may beadapted by the skilled artisan in view of the guidance provided herein.Isolation of VRE-active bacteriophage from suitable samples typicallyproceeds by mixing the sample with nutrient broth, inoculating the brothwith a host bacterial strain, and incubating to enrich the mixture withbacteriophage that can infect the host strain. An Enterococcus sp.strain will be used as the host strain, preferably a VRE strain. Afterthe incubation for enrichment, the mixture is filtered to removebacterial leaving lytic bacteriophage in the filtrate. Serial dilutionsof the filtrate are plated on a lawn of VRE, and VRE-active phage infectand lyse neighboring bacteria. However the agar limits the physicalspread of the phage throughout the plate, resulting in small visiblyclear areas called plaques on the plate where bacteriophage hasdestroyed VRE within the confluent lawn of VRE growth. Since one plaquewith a distinct morphology represents one phage particle that replicatedin VRE within that area of the bacterial lawn, the purity of abacteriophage preparation can be ensured by removing the material inthat plaque with a pasteur pipette (a “plaque pick”) and using thismaterial as the inoculum for further growth cycles of the phage. Thebacteriophage produced in such cycles represent a single strain or“monophage.” The purity of phage preparation (including confirmationthat it is a monophage and not a polyvalent phage preparation) isassessed by a combination of electron microscopy, SDS-PAGE, DNArestriction digest and analytical ultracentrifugation. In addition, eachphage is uniquely identified by its DNA restriction digest profile,protein composition, and/or genome sequence.

Individual VRE-active bacteriophage strains (i.e., monophages) arepropagated as described for enrichment culture above, and then testedfor activity against multiple VRE strains to select broad-spectrumVRE-active bacteriophage. Efforts are made to select phages that (i) arelytic, (ii) are specific to enterococci, (iii) lyse more than 70% of theVRE strains in our VRE strain collection, and/or (iv) lyse VRE strainsresistant to other VRE phages previously identified. It is also possibleto select appropriate phages based upon the sequences of DNA or RNAencoding proteins involved in the binding and/or entry of phage intotheir specific host, or based upon the amino acid sequences or antigenicproperties of such proteins.

Quantities of broad-spectrum VRE-active bacteriophage needed fortherapeutic uses described below may be produced by culture on asuitable host strain in the mariner described above for enrichmentculture. When performing an enrichment culture to produce bacteriophagefor therapeutic use, a host strain is selected based on its ability togive a maximum yield of phage, as determined in pilot experiments withseveral different host VRE strains. If two or more host strains givesimilar yield the strain most sensitive to antibiotics is selected.

The techniques described herein for isolation of VRE monophages areapplicable to isolation of bacteriophages that are lytic for otherpathogenic bacteria. Substitution of host strains of other bacteria willresult in isolation of phage specific for those bacteria. Starting theisolation process with samples that also contain bacteria of the hostspecies will accelerate the process.

Isolation of phage for MDRSA or for resistant Pseudomonas species can beaccomplished by a skilled artisan in a fashion completely analogous tothe isolation of VRE phage.

Patient Population

Any patient who is at risk for colonization with VRE, MDRSA, multi-drugresistant Pseudomonas, or other antibiotic-resistant species, or who hasproven VRE colonization is a candidate for treatment according to themethod of this invention. Intestinal colonization with VRE is relativelycommon in institutionalized patients undergoing antimicrobial therapy.In studies conducted in 1993-94, 17-19% of a random sample of allpatients at the University of Maryland Hospital were colonized with VRE(Morris, et al. (1995), “Enterococci resistant to multiple antimicrobialagents including vancomycin.” Ann. Int. Med., 123:250-9), while in anidentical study conducted in 1996 this increased to 23.8%. Oncecolonized with VRE, a patient may remain colonized for life; howeveronce off antimicrobial therapy, VRE colonization may drop to levels notdetectable in routine stool culture. Colonized persons though who alsosubsequently become immunocompromised are at risk for developingbacteremia (Edmond, et .al., 1995; Tomieporth, et al (1996), “Riskfactors associated with vancomycin resistant Enterococcus faeciumcolonization or infection in 145 matched case patients and controlpatients.” Clin. Infect. Dis., 23:767-72).

VRE infection is a particularly serious problem among immunocompromisedand/or seriously ill patients in cancer centers, intensive care units,and organ transplant centers. In case control studies VRE has beenlinked to antimicrobial use and severity of illness (as measured byAPACHE score) (Handwerger, et al. (1993), “Nosocomial outbreak due toEnterococcus faecium, highly resistant to vancomycin, penicillin andgentamicin.” Clin. Infect. Dis., 16:750-5; Montecalvo, et al. (1996),“Bloodstream infections with vancomycin resistant enterococci.” Arch.Intern. Med., 156:1458-62; Papanicolaou, et al. (1996), “Nosocomialinfections with vancomycin-resistant Enterococcus faecium in livertransplant patients: Risk factors for acquisition and mortality.” Clan.Infect. Dis., 23:760-6; Roghmann, et al., (1997), “Recurrent vancomycinresistant Enterococcus faecium bacteremia in a leukemic patient who waspersistently colonized with vancomycin resistant enterococci for twoyears.” Clin. Infect. Dis., 24;514-5). Investigators at the Universityof Maryland at Baltimore and the Baltimore VA Medical Center havedemonstrated by pulse field electrophoresis that VRE strains causingbacteremia in cancer patients are almost always identical to those thatcolonize the patient's gastrointestinal tract.

Three categories of immunocompromised patients subjected to prolongedantimicrobial administration in a institutionalized setting and whowould be susceptible to VRE gastrointestinal colonization are: 1)leukemia (30,200 patients per year in the U.S.) and lymphoma patients(64,000 patients per year in the U.S.), 2) transplant patients (20,961per year in the U.S.), and 3) AIDS patients (66,659 patients per year inthe U.S.). The total number of patients in the immunocompromisedcategory is 181,800 per year in the U.S. Pfundstein, et al., found thatthe typical rate of enterococcal gastrointestinal colonization amongrenal and pancreas transplant patients receiving antibiotics in aninstitutional setting was 34% (38/102) with 4 (11%) of these isolatesbeing VRE (Pfundstein, et al. (1999), “A randomized trial of surgicalantimicrobial prophylaxis with and without vancomycin in organtransplant patients.” Clin. Transplant., 13:245-52). Therefore the rateof gastrointestinal colonization by VRE in this immunocompromisedpopulation would be 0.34×0.11=0.04 or 4% of the total patientpopulation. One can therefore estimate VRE gastrointestinal,colonization to be 181,800×0.04=7272 patients per year.

Formulation and Therapy

According to this invention, VRE-active bacteriophage are preferablyformulated in pharmaceutical compositions containing the bacteriophageand a pharmaceutically acceptable carrier, and can be stored as aconcentrated aqueous solution or lyophilized powder preparation.Bacteriophage may be formulated for oral administration by resuspendingpurified phage preparation in aqueous medium, such as deionized water,mineral water, 5% sucrose solution, glycerol, dextran, polyethyleneglycol, sorbitol, or such other formulations that maintain phageviability, and are non-toxic to humans. The pharmaceutical compositionmay contain other components so long as the other components do notreduce the effectiveness (ineffectivity) of the bacteriophage so muchthat the therapy is negated. Pharmaceutically acceptable carriers arewell known, and one skilled in the pharmaceutical art can easily selectcarriers suitable for particular routes of administration (Remington'sPharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985).

The pharmaceutical compositions containing VRE-active bacteriophage maybe administered by parenteral (subcutaneously, intramuscularly,intravenously, intraperitoneally, intrapleurally, intravesicularly orintrathecally), topical, oral, rectal, inhalation, ocular, otic, ornasal route, as necessitated by choice of drug and disease.

Injection of specific lytic phages directly into the bloodstream caneliminate or significantly reduce the number of targeted bacteria in theblood. If, after either oral or local administration, phages get intothe bloodstream in sufficient numbers to eliminate bacteria from thebloodstream, septicemia may be treated by administering phages orally(or locally). If the phages do not get into the bloodstream insufficient numbers to eliminate bacteria from the bloodstream, theutility of direct i.v. injection of phages for treating septicinfections can be used to treat bloodstream infections caused by VRE andother pathogenic bacteria, and can provide an urgently needed means fordealing with currently untreatable septicemic infections.

Dose and duration of therapy will depend on a variety of factors,including the patient age, patient weight, and tolerance of the page.Bacteriophage may be administered to patients in need of the therapyprovided by this invention by oral administration. Based on previoushuman experience in Europe, a dose of phage between 10⁷ and 10¹¹ PFUwill be suitable in most instances. The phage may be administered orallyin, for example, mineral water, optionally with 2.0 grams of sodiumbicarbonate added to reduce stomach acidity. Alternatively, sodiumbicarbonate may be administered separately to the patient just prior todosing with the phage. Phages also may be incorporated in a tablet orcapsule which will enable transfer of phages through the stomach with noreduction of phage viability due to gastric acidity, and release offully active phages in the small intestine. The frequency of dosing willvary depending on how well the phage is tolerated by the patient and howeffective a single versus multiple dose is at reducing VREgastrointestinal colonization.

The dose of VRE-active bacteriophage and duration of therapy for aparticular patient can be determined by the skilled clinician usingstandard pharmacological approaches in view of the above factors. Theresponse to treatment may be monitored by, analysis of blood or bodyfluid levels of VRE, or VRE levels in relevant tissues or monitoringdisease state in the patient. The skilled clinician will adjust the doseand duration of therapy based ors the response to treatment revealed bythese measurements.

One of the major concerns about the use of phages in clinical settingsis the possible development of bacterial resistance against them.However, as with antimicrobial resistance, the development of resistanceto phages takes time. The successful use of phages in clinical settingswill require continual monitoring for the development of resistance,and, when resistance appears, the substitution of other phages to whichthe bacterial mutants are not resistant. In general, phage preparationsmay be constructed by mixing several separately grown andwell-characterized lytic monophages, in order to (i) achieve thedesired, broad target activity of the phage preparation, (ii) ensurethat the preparation has stable lytic properties, and (iii) minimize thedevelopment of resistance against the preparation.

The development of neutralizing antibodies against a specific phage alsois possible, especially after parenteral administration (it is less of aconcern when phages are administered orally and/or locally). However,the development of neutralizing antibodies may not pose a significantobstacle in the proposed clinical settings, because the kinetics ofphage action is much faster than is the host production of neutralizingantibodies. For VRE for example, phages will be used for just a fewdays, sufficient to reduce VRE colonization during the time period whenimmunocompromised patients are most susceptible to the development ofpotentially fatal VRE septicemia, but not long enough forphage-neutralizing antibodies to develop. If the development ofantiphage antibodies is a problem, several strategies can be used toaddress this issue. For example, different phages having the samespectrum of activity (but a different antigenic profile) may beadministered at different times during the course of therapy. On a moresophisticated level, therapeutic phages may be genetically engineeredwhich will have a broad lytic range and/or be less immunogenic in humansand animals.

Environmental Therapy

In the 1980's a number of British studies were conducted whichdemonstrated the efficacy of bacteriophage prophylaxis and therapy inmice and farm animal models. These studies were significant because thetiters of the phage preparations administered were significantly lessthan the bacterial inoculum indicating in vivo bacteriophagemultiplication. For example, Smith et al (Smith, et al. (1982),“Successful treatment of experimental Escherichia coli infections inmice using phage: its general superiority over antibiotics.” J. Gen.Microbiol., 128:307-1825) found intramuscular inoculation of mice with10⁶ CFU of E. coli with K1 capsule killed 10/10 mice. However when micewere simultaneously intramuscularly inoculated with 10⁴ PFU of phage, ata separate site, 10/10 mice survived. Smith and coworkers demonstratedthat administration of a mixture of two phage resulted in high levels ofprotection of calves with diarrhea induced by E. coli with K 88 or K99fimbriae (Smith, et al. (1983), “Effectiveness of phages in treatingexperimental Escherichia coli diarrhea in calves, piglets and lambs.” J.Gen. Microbiol., 129:2659-75; Smith, et al. (1987), “The control ofexperimental Escherichia coli diarrhea in calves by means ofbacteriophage.” J. Gen. Microbiol., 133:1111-26; Smith, et al. (1987),“Factors influencing the survival and multiplication of bacteriophagesin calves and in their environment.” J. Gen. Microbiol., 133:1127-35).If the phage was administered before or at tire same time as E. coli nodeaths occurred and complete protection was attained. Control animalsdeveloped watery diarrhea and died within 2 to 5 days. If phageadministration was delayed until the onset of diarrhea, protection wasnot complete although the severity of infection was greatly reduced andno deaths were observed. Berchieri, et al., found that fewer chicksorally infected with 10⁹ PFU of Salmonella typhimurium died when 10⁹ PFUof Salmonella specific phage was orally administered soon afterinitiation of the bacterial infection (Berchieri, et al. (1991), “Theactivity in the chicken alimentary tract of bacteriophages lytic forSalmonella typhimurium.” Res. Microbiol., 142:541-49). They also foundthat the phage was readily spread between the different infected birds.

Environmental applications of phage in health care institutions couldlie most useful for equipment such as endoscopes and environments suchas ICUs which maybe potential sources of nosocomial infection due topathogens such as VRE but which may be difficult or impossible todisinfect. Phage would be particularly useful in treating equipment orenvironments inhabited by bacterial genera such as Pseudomonas which maybecome resistant to commonly used disinfectants. In the Soviet Unionthere has been a report that application of phage to the hospitalenvironment has resulted in killing targeted bacteria such asStaphylococci and Pseudomonas within 48-72 hours. Phage persisted in theenvironment as long as there were target bacteria present and uponelimination of target bacteria, phage became undetectable in 6-8 days(Alavidze, et al, 1988, “Use of specific bacteriophage in theprophylaxis of intrahospital infections caused by P. aeruginosa.” inAbstracts, All-Soviet Union conference “Modern biology at the service ofpublic health”. Kiev, Ukraine).

Phage compositions used to disinfect inanimate objects or theenvironment may be sprayed, painted, or poured, onto such objects orsurfaces in aqueous solutions with phage titers ranging between 10⁷-10¹¹PFU/ml. Alternatively, phage may be applied by aerosolizing agents thatmight include dry dispersants which would facilitate distribution of thephage into the environment. Such agents may also be included in thespray if compatible with phage viability and nontoxic in nature.Finally, objects may be immersed in a solution containing phage. Theoptimal numbers and timing of applications of phage compositions remainsto be determined and would be predicated by the exact usage of suchproducts.

Since phage are normally widely present in the environment and are foundeven in food or drugs, there is minimal safety concern with regard toapplying phage preparations to the environment.

As reported above, Smith and Huggins in England found that E. coliinduced diarrhea in calves could be prevented by simply spraying thelitter in the calf rooms with an aqueous phage preparation or even bykeeping the calves in uncleaned rooms previously occupied by calveswhose E. coli infections had been treated with phage. There is also datafrom the Soviet Union indicating the efficacy of phage to rid chickenhouses of Staphylococci (Ponomarchuk, et al., (1987), “Strain phageStaphylococci applicable for prophylaxis and therapy of poultryStaphylococcus.” Soviet patent N1389287, Dec. 15, 1987).

In the future, application of VRE phage to the environment of farmanimals such as chickens or cattle maybe necessary to reduce VRE in thissetting if VRE become prevalent in such environments and such animal VREare capable, upon being consumed ire contaminated food, of transientlycolonizing the human gastrointestinal tract long enough to transferantibiotic resistance gene transposons to normal gut flora (Latta, S.(1999) “Debate heats up over antibiotic-resistant foodborne bacteria.”The Scientist 13; (14) 4-5).

Bacteriophage Cocktails

This invention also contemplates phage cocktails which may be customtailored to the pathogens that are prevalent in a certain situation.Typically, pathogenic bacteria would be initially isolated from aparticular source (e.g., a patient or location contaminated with VRE)and susceptibility testing of the pathogens to various bacteriophagestrains would be performed, analogous to antimicrobial susceptibilitytesting. Once each pathogen's phage susceptibility profile isdetermined, the appropriate phage cocktail can be formulated from phagestrains to which the pathogens are susceptible and administered to thepatient. Since phage would often be used in institutional settings wherepathogens are resistant to many antimicrobial agents, phage cocktailswould often consist of phage lytic for the most prevalent institutionalpathogens which, in addition to enterococci, are Staphylococcus aureus,Staphylococcus epidermidis, E. coli and Pseudomonas aeruginosa. Alsosince enterococci are often involved in polymicrobial infections alongwith other gastrointestinal commensals, such as in pelvic woundinfections, the approach of therapeutically using cocktails of phagelytic against different bacterial species would be most appropriate.Since phage cocktails would be constructed of phage againstinstitutional pathogens, isolation of such phage would be mostsuccessful from the sewage of such institutions. Typically, the phagecocktail will include one or more VRE-active bacteriophage according tothis invention.

It may be appropriate to use certain phage cocktails in agriculturalsettings where there are certain human pathogens such as Salmonella andCampylobacter inherent to poultry or livestock and which contaminate theenvironment of such animals on an ongoing basis. The result is acontinuing source of infection by such pathogens.

Bacteriophage cocktails may be applied contemporaneously—that is, theymay be applied at the same time (e.g., in the same application), or maybe applied in separate applications spaced in time such that they areeffective at the same time. The bacteriophage may be applied as a singleapplication, periodic applications, or as a continuous application.

Other bacteria within the contemplation of the present inventioninclude, inter alia, Campylobacter, E. coli H7:O157, and Listeria, andStapholocoocus.

Bacteriophages as Sanitation Agents

Phages may be used as sanitation agents in a variety of fields. Althoughthe terms “phage” or “bacteriophage” may be used below, it should benoted that, where appropriate, this term should be broadly construed toinclude a single bacteriophage, multiple bacteriophages, such as abacteriophage cocktail, and mixtures of a bacteriophage with an agent,such as a disinfectant, a detergent, a surfactant, water, etc.

The efficacy of phage treatment to reduce bacterial load may bedetermined by quantitating bacteria periodically in samples taken fromthe treated environment. In one embodiment, this may be performed daily.If administration of phage reduced bacterial load by at least 1 log ascompared to the control (e.g., before treatment) within 48-98 hoursafter phage administration, then this dose of the particular phage isdeemed efficacious. More preferably, colonization will be reduced by atleast 3 logs.

Applications

According to some embodiments of the present invention, bacteriophagesmay be used for food and agriculture sanitation (including meats, fruitsand vegetable sanitation), hospital sanitation, home sanitation,military sanitation (including anti-bioterrorism applications andmilitary vehicle and equipment sanitation), industrial sanitation, etc.Other applications not specifically mentioned are within thecontemplation of the present invention.

1. Food and Agriculture Sanitation

The broad concept of bacteriophage sanitation may be applied to otheragricultural applications and organisms. Produce, including fruits andvegetables, dairy products, and other agricultural products consumed byhumans may become contaminated with many pathogenic organisms, includingSalmonella and highly virulent organisms such as E. coli O157:H7. Forexample, freshly-cut produce frequently arrive at the processing plantcontaminated with pathogenic bacteria at concentrations ranging from 10⁴to 10⁶ colony forming units (CFU) per gram of food. Salmonellaenteritidis is able to survive and grow on fresh-cut produce underconditions mimicking “real life” settings, and fresh-cut fruits having aless acidic pH (e.g., a pH of about 5.8; such as honeydew melons) areespecially prone to becoming overgrown with Salmonella.

A significant proportion of produce consumed in the United Statesoriginates in countries lacking the high sanitation standards of theUnited States. In the past, this has led to outbreaks of food-borneillness traceable to imported produce. The application of bacteriophagepreparations to agricultural produce can substantially reduce oreliminate the possibility of food-borne illness through application of asingle phage or phage cocktails with specificity toward species ofbacteria associated with food-borne illness. Bacteriophage may beapplied at various stages of production and processing to reducebacterial contamination at that point or to protect againstcontamination at subsequent points.

During the studies performed by the inventors in collaboration withIntralytix, Inc., it has been shown that the SCLPX phage mixture reducesthe numbers of Salmonella on honeydew melon slices by approximately 3.5log units (see Example 7). This level of reduction is significantlyhigher than the maximum reduction rate of 1.3 logs in bacterial countsreported for fresh-cut fruits using the most effective chemicalsanitizer (hydrogen peroxide). See Liao, C. H. and G. M. Sapers“Attachment and growth of Salmonella Chester on apple fruits and in vivoresponse of attached bacteria to sanitizer treatments” J. Food Prot.63:876-83 (2000); Beuchat, Nail, et al. 1998 1003. However, because somephages may have difficulty in withstanding acidic pH, the treatment maynot be as effective on produce with an acidic pH, such as Red Deliciousapples. With high pH produce, in one embodiment, higher concentrationsof phages may be applied to the produce. In another embodiment, theadministration of the phages to the produce may be repeated. In stillanother embodiment, pH-resistant phage mutants may be selected andapplied to the highly acidic produce.

The use of specific phages as biocontrol agents on produce provides manyadvantages. Examples include the facts that phages are natural,non-toxic products that will not disturb the ecological balance of thenatural microflora in the way the common chemical sanitizers do, butwill specifically lyse the targeted food-borne pathogens. In thiscontext, the SCLPX mixture is only effective against Salmonellae, andgenerally does not lyse other bacteria, such as E. coli, S. aureus, P.aeruginosa, Lactobacillus, Streptococcus, and enterococci. Shouldadditional coverage be required, phages lytic for more than one pathogencan be combined and used to target several pathogenic bacteriasimultaneously.

Phages also provide additional flexibility for long-term applications.For example, it has been reported that many bacteria are developingresistance to sanitizers commonly used in the fresh-cut produceindustry. See Chesney, J. A., J. W. Eaton, and J. R. JR. Mahoney,“Bacterial Glutathione: a Sacrificial Defense against ChlorineCompounds” Journal of Bacteriology 178:2131-35 (1996); Mokgatla, R. M.,V. S. Brözel, and P. A. Gouws “Isolation of Salmonella Resistant ToHypochlorous Acid From A Poultry Abattoir” Letters in AppliedMicrobiology 27:379-382 (1998). Although it is likely that resistancewill also eventually develop against certain phages, there are importantdifferences between phages and chemical sanitizers that favor the use ofphages as biocontrol agents. For example, the development of resistanceagainst phages can be reduced by constructing and using a cocktail ofphages containing several lytic phages (similar to the SCLPXpreparation), so that when the bacteria develop resistance to one phagein the preparation, the resistant mutants will be lysed by other phagesand will not be able to propagate and spread further. Furthermore,because phages, unlike chemical sanitizers, are natural products thatevolve along with their host bacteria, new phages that are activeagainst recently emerged, resistant bacteria can be rapidly identifiedwhen required, whereas identification of a new effective sanitizer is amuch longer process which may take several years.

In one embodiment, the use of specific bacteriophages, in addition towashing of fresh-cut produce with water and keeping the produce at lowtemperatures (approximately 50° C.), provides an efficient method forpreventing food-borne human pathogens, like Salmonella, from growing andbecoming a health hazard on at least some produce, includingfreshly-cut, damaged, diseased, and healthy produce.

Specific bacteriophages may be applied to produce in restaurants,grocery stores, produce distribution centers, etc. For example, phagemay be periodically or continuously applied to the fruit and vegetablecontents of a salad bar. This may be though a misting or sprayingprocess, washing process, etc., and may be provided as a substitute orsupplement to chemical sanitizers, such as hypochlorite, sulfur dioxide,etc.

In another embodiment, phage may be periodically or continuously appliedto produce in a grocery store. In still another embodiment, phage may beapplied to produce in produce distribution centers, in shipmentvehicles, etc. Other applications are within the contemplation of thepresent invention.

A bacteriocin may also be applied to the produce. In one embodiment,bacteriocin nisin, which is sold under the name Nisaplin®, and availablefrom Aplin & Barrett Ltd, Clarks Mill, Stallard Street, Trowbridge,Wilts BA14 8HH, UK, may be used. Nisin is produced by Lactococcusstrains, and has been used to control bacterial spoilage in bothheat-processed and low-pH foods. Nisin is active against Listeriamonocytogenes, especially at low pH, which complements the phageapplication.

Another embodiment of this application contemplates inclusion ofbacteriophage or matrices or support media containing bacteriophageswith packaging containing meat, produce, cut fruits and vegetables, andother foodstuffs. Bacteriophage preparations containing singlebacteriophages or cocktails of bacteriophages specific for the desiredpathogen(s) may be sprayed, coated, etc. onto the foodstuff or packagingmaterial prior to packaging. The bacteriophage preparation may also beintroduced into the package as part of a matrix that may releaseadsorbed or otherwise incorporated phage at a desirable rate by passivemeans, or may comprise part of a biodegradable matrix designed torelease phage at a desirable rate as it degrades. Examples of passiverelease devices may include absorbent pads made of paper or otherfibrous material, sponge, or plastic materials.

In another embodiment, a polymer that is suitable for packaging may beimpregnated with a bacteriophage preparation. A suitable method forimpregnating a polymer with a bacteriophage preparation is disclosed inU.S. Pat. No. 60/175,377, which is incorporated by reference in itsentirety. Suitable polymers may include those polymers approved by theU.S. Food and Drug Administration for food packaging.

In another embodiment, bacteriophage preparations specific forClostridium botulinum may be a desirable means of preventing botulism infoodstuffs such as bacon, ham, smoked meats, smoked fish, and sausages.Present technology requires high concentrations of nitrates and nitritesin order to meet the United States Government standard for C. botulinum.Bacteriophage preparations would permit reduction or possibleelimination of these potentially carcinogenic substances. Methods ofapplication include spraying as an aerosol, application of liquid to thesurface with a spreading device, injection of a liquid, or incorporationof a liquid bacteriophage preparation into products requiring mixing.

2. Hospital Sanitation

Bacteriophages may be used to sanitize hospital facilities, includingoperating rooms, patient rooms, waiting rooms, lab rooms, or othermiscellaneous hospital equipment. This equipment may includeelectrocardiographs, respirators, cardiovascular assist devices,intraaortic balloon pumps, infusion devices, other patient care devices,televisions, monitors, remote controls, telephones, beds, etc. Thepresent invention provides a fast and easy way to sanitize certainsensitive equipment and devices.

In some situations, it may be desirable to apply the phage through anaerosol canister; in other situations, it may be desirable to wipe thephage on the object with a transfer vehicle; in still other situations,it may be desirable to immerse the object in a container containingphages; and in others, a combination of methods, devices, or techniquesmay be used. Any other suitable technique or method may be used to applythe phage to the area, object, or equipment.

Phages may be used in conjunction with patient care devices. In oneembodiment, phage may be used in conjunction with a conventionalventilator or respiratory therapy device to clean the internal andexternal surfaces between patients. Examples of ventilators includedevices to support ventilation during surgery, devices to supportventilation of incapacitated patients, and similar equipment. This mayinclude automatic or motorized devices, or manual bag-type devices suchas are commonly found in emergency rooms and ambulances. Respiratorytherapy devices may include inhalers to introduce medications such asbronchodilators as commonly used with chronic obstructive pulmonarydisease or asthma, or devices to maintain airway patency such ascontinuous positive airway pressure devices.

In another embodiment, phage may be used to cleanse surfaces and treatcolonized people in an area where highly-contagious bacterial diseases,such as meningitis or enteric infections such as those caused byShigella species have been identified. Bacterial meningitis, such asmeningitis caused by Neisseria meningitides frequently occurs insettings where children or young adults are closely clustered such asschools, dormitories, and military barracks. The pathogen is spread asan aerosol. Shigella is commonly spread through fecal-oral transmission,where the spread may be direct, or may be through intermediarycontaminated surfaces or food or water. Bacterial pathogens spread as anaerosol may be treated through introduction of bacteriophage into theenvironment as an aerosol continuously or episodically. Bacterialinfections spread through contact with contaminated surfaces may betreated with appliances to distribute bacteriophage-containingpreparations into those surfaces. Contaminated water, most specificallycontaminated water supplies such as cisterns, wells, reservoirs, holdingtanks, aqueducts, conduits, and similar water distribution devices maybe treated by introduction of bacteriophage preparations capable oflysing the intended pathogen.

3. Home and Public Area Sanitation

In another embodiment, bacteriophages may be used to sanitize a livingarea, such as a house, apartment, condominium, dormitory, barracks, etc.The phage may also be used to sanitize public areas, such as theaters,concert halls, museums, train stations, airports , etc.

The phage may be dispensed from conventional devices, including pumpsprayers, aerosol containers, squirt bottles, pre-moistened towelettes,etc. The phage may be applied directly to (e.g., sprayed onto) the areato be sanitized, or it may be transferred to the area via a transfervehicle, such as a towel, sponge, etc.

Phage may be applied to various rooms of a house, including the kitchen,bedrooms, bathrooms, garage, basement, etc. In embodiment, the phage maybe used in the same manner as conventional cleaners (e.g., Lysol®cleaner, 409® cleaner, etc.).

In one embodiment, phage may be applied in conjunction with (before,after, or simultaneously with) conventional cleaners provided that theconventional cleaner is formulated so as to preserve adequatebacteriophage biologic activity.

In one embodiment, phage may be used to sanitize pet areas, such as petbeds, litter boxes, etc.

4. Military Applications

Bacteriophages may be used to decontaminate military equipment. In oneembodiment, this may include decontaminating vehicles, aircraft,weapons, miscellaneous soldier equipment, etc. that have beencontaminated by biological weapons or agents, such as Anthrax. Aircraftand other equipment with sensitive outer surfaces, such as stealthaircraft, or sensitive electronics located on or near those surfaces,may be damaged, or destroyed, by the application of knowndecontamination fluids or techniques. Thus, this damage may be avoidedby using bacteriophages to decontaminate these surfaces.

In one embodiment, the phage may be sprayed on the equipment by hoses orother spraying devices. In another embodiment, a “car wash” may beconstructed to coat a vehicle with phages as the vehicle passes throughthe “car wash.” Other methods, apparatuses, techniques, and devices arewithin the contemplation of this invention.

Bacteriophages may also be used to combat bioterrorism and biologicwarfare, which is defined as the intentional introduction of pathogenicbacteria into the environment by means where it is likely to infecthuman populations and cause disease. Bioterrorism may includeintroduction of pathogenic bacteria into buildings, vehicles, foodsupplies, water supplies, or other similar settings. Biologic warfaremay involve dispersal of pathogenic bacteria by missiles, explosivedevices, aircraft, ships, and other similar devices in ways likely toinfect targeted populations or individuals.

In one embodiment, bacteriophage may be used to decontaminate largeobjects, including the interior and exterior of buildings. Here, thephage may be sprayed or otherwise applied to contaminated surfaces. Inanother embodiment, the phage may be used to decontaminate large areasof land. For example, the phage may be applied by crop sprayers (e.g.,both fixed-wing and rotary wing aircraft), by irrigation sprinklers, orby any suitable means.

Where appropriate, the application of a bacteriophage cocktail is withinthe contemplation of the present invention.

5. Industrial Applications

The present invention may be used in many industrial applications,including the animal husbandry industry. This includes, but is notlimited to, the breeding, raising, storing, and slaughter of livestockor other animals.

Referring to FIG. 1, an example of how to use bacteriophage in a poultryprocessing plant is provided. It should be recognized that phages may beapplied at any stage; the preferred locations for the phage applicationare identified in this figure. Although the word “spray” may be used inconjunction with the description below, it should be recognized thatrinsing (e.g., in a washing tank) and providing phages as a food or adrinking additive (e.g., mixing the phages with food or water, or both),where appropriate, may be substituted, or used in conjunction withspraying.

After the fertilized eggs are collected in the Fertilized Egg CollectionSite, the fertilized eggs may be sprayed with phages before they aretransferred to incubators in the hatchery (A). It has not been possibleto consistently eliminate Salmonella from breeder flocks, and,consequently, Salmonella may be present on the surface of fertilizedeggs; conditions in incubators promote multiplication of the organism,and chicks may become infected as they peck out of the egg. Aggressivewashing of eggs and the use of disinfectants of sufficient strength toeliminate all bacterial contamination is not desirable with fertilizedeggs. In this setting, spraying phages onto the surface of the eggs mayprovide a means of minimizing Salmonella contamination of hatchedchicks.

After the birds are hatched, the birds may be sprayed with phages beforethey are transferred to a chicken house or to a farm (B). Immediatelyafter hatching, chicks may be sprayed with various viral vaccines(Newcastle, bronchitis, INDIA) which are ingested as the animals preentheir feathers. A small percentage of chicks are Salmonella-positive atthis point in time (see comments above about Salmonella on eggs);however, once introduced into chicken houses, contamination may spreadrapidly to all animals in the house. Application of phage immediatelyafter hatching and before transfer to chicken houses may reduce the riskof the bacterium being spread from the chicks to the rest of the birdsin the chicken house.

During raising in the chicken house or farm, the birds may be providedwith phages in their drinking water, food, or both (C). Once mature, thebirds are transferred to the slaughter area, where they are slaughtered,and then transferred to a washing area, where they are processed andwashed. Phages may be sprayed onto the chicken carcasses after thechlorine wash in chiller tanks, before post-chill processing (D).Salmonella contamination at this point should be minimized, andapplication of phages may provide a “final product clean-up.” Inaddition, only a small amount of phage preparation will be needed(approximately 5-10 ml per chicken) instead of several hundred litersrequired to decontaminate a chicken house. Another advantage of applyingphages at this stage is that since phages will not be carried to lociwhere they can readily be exposed to Salmonella for a long period oftime (e.g., to a chicken house), the risk of Salmonella developingresistance against the phage(s) will be greatly reduced.

After slaughter, the birds are chilled. The chilled birds are thenprocessed, which may include sorting, cutting the birds, packaging,etc., and are then transported to designated points of sale.

It is also possible to sanitize the areas that the birds contact. Thisincludes the egg collection site, the incubator/hatchery, the chickenhouse, the slaughter area, and the processing areas, and any equipmentthat is used or contained therein. Similar procedures may be employedfor the reduction of bacterial contamination on eggs produced for saleand/or consumption. In addition to use contemplated for Salmonella, thismethod may be particularly well suited to the decontamination ofenvironmental pathogens, specifically including Listeria monocytogenes.

In one embodiment, the working phage concentration may range from1×10⁵−1×10⁹ PFU/ml.

One of ordinary skill in the art should recognize that the exampleprovided in FIG. 1 is easily adaptable for other species of animals,including calves, pigs, lamb, etc, even if the animals are notslaughtered. For example, the present invention may have applications inzoos, including cages, holding areas, etc.

Where appropriate, the application of a bacteriophage cocktail is withinthe contemplation of the present invention.

In another embodiment, phages may be applied to industrial holdingtanks. For instance, in areas in which products are milled, water, oil,cooling fluids, and other liquids may accumulate in collection pools.Specific phages may be periodically introduced to the collection poolsin order to reduce bacterial growth. This may be through spraying thephage on the surface of the collection pool, wherein it is most likelythat the bacteria may be located, or through adding phage into thecollection pool.

Devices

1. General

According to one embodiment of the present invention, phages may storedin a container, and then applied to an area or an object. The containermay range in size from a small bottle to a large industrial storagetank, which may be mobile or fixed.

The container of the present invention may use a variety of mechanismsto apply the phage to an object. In general, any mechanism that providesa substantially even dispersion of the phage may be used. Further, thephage should be dispersed at a pressure that does not cause substantialdamage to the object to which the phage is being applied, or at apressure that causes damage, directly or indirectly, to the phageitself.

It has been found that some bacteriophages may be inactivated due tointerfacial forces, while other bacteriophages survive such forces.Adams suggested that air-water interface was responsible forbacteriophage inactivation. See Adams, M. H. “Surface inactivation ofbacterial viruses and of proteins” J. Gen. Physiol. 31:417432 (1948)(incorporated by reference in its entirety). In addition, Adams foundthat it is the presence of proteins in the diluent protected the severalcoli-dysentery bacteriophages from inactivation.

Trouwborst et al. conducted several studies on bacteriophageinactivation. See Trouwborst, T., J. C. de Jong, and K. C. Winkler,“Mechanism of inactivation in aerosols of bacteriophage T₁ ” J. Gen.Virol. 15:235-242 (1972); Trouwborst, T., and K. C. Winkler “Protectionagainst aerosol-inactivation of bacteriophage T₁ by peptides and aminoacids” J. Gen. Virol. 17:1-11 (1972); Trouwborst, T., and J. C. de Jong“Interaction of some factors in the mechanism of inactivation ofbacteriophage MS2 in aerosols” Appl. Microbiol. 26:252-257 (1973); andTrouwborst, T., S. Kuyper, J. C. de Jong, and A. D. Plantinga“Inactivation of some bacterial and animal viruses by exposure toliquid-air interfaces” J. Gen. Virol. 24:155-165 (1974), all of whichare incorporated, by reference, in their entireties. In “Mechanism ofinactivation in aerosols of bacteriophage T₁” the data suggested thatsurvival of the bacteriophage T₁ varied with relative humidity, with aminimum survival near the relative humidity corresponding to a saturatedsolution of the salt, and a better survival at a lower initial saltconcentration. The authors found when the T₁ bacteriophage was shaken,or when it was an aerosol, surface inactivation was a major cause ofinactivation. The data suggested, however, that broth protected T₁against aerosol inactivation. Subsequently, in “Protection againstaerosol-inactivation of bacteriophage T₁ by peptides and amino acids,”Trouwborst et al. determined that the phage T₁ may be protected fromaerosol-inactivation by peptone and by apolar amino acids, such asleucine and phenylalanine. In addition, the authors found that peptonealso protects T₁ from inactivation from low relative humidity.

In “Inactivation of some bacterial and animal viruses by exposure toliquid-air interfaces,” Trouwborst et al. subjected the bacteriophagesT₁, T₃; T₅, MS₂, of the EMC virus and of the Semliki Forest virus to alarge air/water interface. The authors determined that the EMC virus wasnot sensitive to this treatment, phage T₃ and T₅ were little affected,and phage T₁ and the Semliki Forest virus were rapidly inactivated. Theauthors also found that inactivation by aeration could be prevented bythe addition of peptone, by apolar carboxylic acids, and by the surfaceactive agent OED. Further, the data suggested that the rate of surfaceinactivation was strongly dependent on the salt concentration of themedium.

In a study conducted by Thompson and Yates (“Bacteriophage Inactivationat the Air-Water-Solid Interface in Dynamic Batch Systems” Applied andEnvironmental Microbiology, 65:1186-1190 (March 1999), which isincorporated by reference in its entirety), three bacteriophages (MS2,R17 and ΦX174) were percolated through tubes containing glass and Teflonbeads. Two of the three phages (MS2 and R17) were inactivated by thisaction, while the third bacteriophage (ΦX174) was not. The datasuggested to the authors that inactivation was dependent upon (1) thepresence of a dynamic air-water-solid interface (where the solid is ahydrophobic surface), (2) the ionic strength of the solution, (3) theconcentration of surface active compounds in the solution, and (4) thetype of virus used.

In addition, in a separate study, Thompson et al. studied the air-waterinterface and its inactivating effect on certain bacteriophages. SeeThompson et al., “Role of the Air-Water-Solid Interface in BacteriophageSorption Experiments”, Applied and Environmental Microbiology,64:304-309 (January 1998) (which is incorporated by reference in itsentirety). In this study, it was observed that the bacteriophage MS2 wasinactivated in control tubes made of polypropylene, while there was nosubstantial inactivation of MS2 in glass tubes. In contrast, thebacteriophage ΦX174 did not undergo inactivation in either polypropyleneor glass tubes. This data suggested that the inactivation of MS2 was dueto the influence of air-water interfacial forces, while ΦX174 was notaffected by the same forces that inactivated MS2.

At least one study has been directed at the type, characteristics, and,properties of membrane. See Mix, T. W. “The physical chemistry ofmembrane-virus interactions” Dev. Ind. Microbiol. 15:136142 (1974)(incorporated by reference in its entirety). Mix identified severalfactors to be considered when determining whether a virus will adsorbonto a membrane, including the nature of the membrane and the virussurfaces, electrostatic forces, environmental factors (pH, the presenceof electrolytes, the presence of competitive adsorbents, temperature,flow rate, etc.). The importance of the factors may vary for differentviruses.

The devices discussed below may be appropriate for most bacteriophages;however, it may be possible to enhance delivery of specificbacteriophages by selecting for phages that are stable in specificdevices before they are used for the indicated purposes. In addition, itmay be beneficial to use different materials (e.g., glass versuspolypropylene) depending on the particular bacteriophage. For example,the studies above suggest that the phage ΦX174 would be effective ifdispensed from through a polypropylene tube and a sprayer, such that aplurality of drops of the phage were formed, while the studies suggestthat the phage MS2 would not be effective in this application regime.Therefore, appropriate devices, materials, and phages should beselected.

In some embodiments, the phage may be maintained under controlledconditions in order to maintain the activity level of the phage, such asin an aqueous or a non-aqueous solution, a gel, etc. In anotherembodiment, the phage may be stored in a freeze-dried state, and may bemixed with a liquid vehicle shortly before use. Suitable vehiclesinclude water, chloroform, and mixtures thereof. Other vehicles includewater containing biologically compatible solutes such as salts andbuffering agents as are commonly known in the art. Such salts andbuffering agents may also consist of volatile solutes, such as ammoniumchloride, or may be non-volatile, such as sodium chloride. Thisembodiment is expressly intended to include all combinations andmixtures of aqueous and organic solvents and solutes that maintainadequate phage viability, which may be greater than 50% of the originaltiter, more preferably greater than 75% of the original titer, or mostpreferably greater than 95% of the original titer.

In another embodiment, the phage may be maintained at a controlledtemperature. In another embodiment, the phage may be maintained at acontrolled pressure.

2. Specific Devices

In one embodiment, a simple manual spray mechanism may be used. In thisdevice, the pressure is generated by the user when the user depressesthe pump (or, if a trigger pump, when the user pulls the “trigger”),causing the phage and its carrier to be forced through the nozzle of themechanism. In another embodiment, the phage may be stored under pressurein an canister, and may be delivered in a conventional manner bydepressing a button, or a valve, on top of the canister. In anotherembodiment, a fogger or misting device may be used to disperse the phageover an area.

In addition to manual sprayers, power sprayers may be used to apply thephage. Example of a suitable sprayer includes the Power Painter, theAmSpray® Double Spray Piston Pump, the High Volume Low Pressure pumps,and the Diaphragm pumps, available from Wagner Spraytech Corporation,Minneapolis, Minn. Other power sprayers, including those much largerthan those listed above, are within the contemplation of the presentinvention.

In another embodiment, rollers, such as a paint roller, may be used.This may include thin film applicators. Within the contemplation of thepresent invention are roller devices, including a roller deviceconnected to a supply of phage that is forced through the roller onto asurface.

Power rollers may also be used. For example, the Wagner® Power Rolleravailable from Wagner Spraytech Corporation, Minneapolis, Minn. may beused. Other power rollers are also within the contemplation of thepresent invention.

For larger applications, hoses, sprayers, sprinklers, or other suitabledevices may be used to apply the phage to the area or to the object fromthe container.

The phage may also be applied manually. For example, the phage may beapplied to the object with a brush. In another embodiment, a transfervehicle, such as a cloth wipe, a paper wipe, a towel, a towelette, asponge, etc. may be used to apply the phage to the object. The transfervehicle may be wiped across an area, or an object, to apply the phage tothe area or object. In one embodiment, the transfer vehicle may beprepackaged, similar to an alcohol wipe.

As discussed above, the phage may be stored in its freeze-dried form,and then combined with the solvent shortly before use. In on embodiment,a package with a glass ampoule containing a solvent may include amaterial coated with the phage in freeze-dried form. When a user wishesto use the phage, the user crushes the ampoule, causing the solvent tomix with the phage. Other technologies for storing the phage and solventseparately, and causing their mixture shortly before use, arewell-known, and may also be used.

In another embodiment, a device that maintains the activity of the phagemay be used. For example, a device that is similar to a fireextinguisher or hand-held plant sprayer may be used to store at leastone bacteriophage under a temperature and pressure that is sufficient tomaintain the activity of the phage(s). This may include providing atemperature control device in order to maintain the temperature, whichmay be powered by A/C current, batteries, etc.

The device may be portable, such that it may be taken to decontaminationsites, or stored in decontamination chambers, etc.

In one embodiment, the phage may have a predetermined “shelf-life,” andmay be periodically changed. In one embodiment, the device may include asensor that warns when the activity level of the phage reaches apredetermined level.

In another embodiment, multiple compartments may be provided formultiple phages, which may be mixed before dispersal from the device.Compartments for at least one agent, such as water, foams,disinfectants, and other agents may be provided, and may also be mixedwith the phage(s) before dispersal, or may be dispersed separately.

The phage may also be maintained in gels and foams. Thus, devices thatdispense gels or foams may be used.

EXAMPLES Example 1 Obtaining VRE Isolates

Isolation of VRE

VRE were isolated by standard methods from patients in the surgicalintensive care and intermediate care units of the University of MarylandMedical Center in Baltimore. Trypticase Soy Agar supplemented with 5%sheep blood (BBL, Cockeysville, Md.) was used to isolate enterococcifrom urine, wounds and sterile body fluids. VRE were isolated from stoolspecimens on Colistin Nalidixic Acid (CNA) agar (Difco labs, Detroit,Mich.) supplemented with defibrinated sheep blood (5%), vancomycin (10μg/ml) and amphotericin (1 μg/ml). See Facklam, R. R., and D. F. Sahm.1995. Enterococcus. In: Manual of Clinical Microbiology, 6^(th) edition,American Society for Microbiology, Washington, D.C., pp. 308-312.

Identification of VRE

Enterococci were identified by esculin hydrolysis and growth in 6.5%NaCl at 45° C. Identification to the species level was done usingconventional testing as indicated in Facklam and Collins (Facklam, etal. (1989), “Identification of Enterococcus species isolated from humaninfections by a conventional method test scheme.” J. Clin. Microbiol.,27:731-4).

Antimicrobial Susceptibility Testing of VRE

Antimicrobial susceptibilities to ampicillin, vancomycin, streptomycin,and gentamicin were determined using the E test quantitative minimuminhibitory concentration procedure (AB Biodisk, Solna Sweden). Qualitycontrol stains of E. faecium (ATCC 29212, 51299) were used to ensurepotency of each antimicrobial agent tested. With exception ofvancomycin, susceptibility interpretations from the National Committeefor Clinical Laboratory Standards were adhered to (National Committeefor Clinical Laboratory Procedures (1993), “Methods for DilutionAntimicrobial Susceptibility Tests for Bacteria that Grow Aerobically.”3rd Edition. National Committee for Clinical Laboratory StandardsVillanova, Pa.; National Committee for Clinical Laboratory Standards(1993), “Performance Standards for Antimicrobial Disk SusceptibilityTests” 5th Edition, National Committee for Clinical LaboratoryStandards, Villanova, Pa.). A VRE isolate was defined as one that had aminimum inhibitory concentration to vancomycin of at least 16 μg/ml;

Defining Generically Distinct VRE Strains

Distinct VRE isolates were characterized as such by contour-clampedhomogeneous electric field electrophoresis after digestion ofchromosomal DNA with SmaI (Verma, P. et al. (1994) “Epidemiologiccharacterization of vancomycin resistant enterococci recovered from aUniversity Hospital” (Abstract). In; Abstracts of the 94th GeneralMeeting of the American Society for Microbiology, Las Vegas, Nev.; Dean,et al. (1994) “Vancomycin resistant enterococci (VRE) of the vanBgenotype demonstrating glycoprotein (G) resistance inducible byvancomycin (V) or teicoplanin (T)” In; Abstracts of the 94th GeneralMeeting of the American Society for Microbiology, Las Vegas, Nev.).Electrophoretic studies were also performed using ApaI digestion for VREstrains which differed only by 1-3 bands after initial analysis(Donabedian, S. M. et al (1992) “Molecular typing ofampicillin-resistant, non-beta lactamase producing Enterococcus faeciumisolates from diverse geographic areas.” J. Clin. Microbiol., 30;2757-61). The vancomycin-resistant genotype (vanA, vanB or vanC) wasdefined by polymerase chain reaction analysis using specific primersselected from published gene sequences (Goering, R. V. and the MolecularEpidemiological Study Group (1994) “Guidelines for evaluating pulsedfield restriction fragment patterns in the epidemiological analysis ofnosocomial infections.” (Abstract) Third International Meeting ofBacterial Epidemiological Markers; Cambridge England).

Example 2 Isolation of VRE Phage

500 ml of raw sewage from the University of Maryland is mixed with 100ml of 10 times concentrated LB broth (Difco Laboratories). Thissewage-broth mixture is inoculated with a 18-24 hour LB broth culture (1ml) of a VRE strain and incubated at 37° C. for 24 hours to enrich themixture for bacteriophage which can infect the VRE strain added. Afterincubation, the mixture is centrifuged at 5000 g for 15 minutes toeliminate matter which may interfere with subsequent filtration. Thesupernatant is filtered through a 0.45 μm Millipore filter. Filtrate isassayed using the Streak Plate Method and/or Appelman Tube TurbidityTest to detect lytic activity against different strains of VRE.

Method for Testing Phage Against VRE Isolates

Three methods are employed: Plaque Assay; Streak Plate Method; and TubeTurbidity Method, and the procedures for each follow.

Plaque Assay:

A 18-24 hour nutrient broth culture of the VILE strain (0.1 ml) to betested for susceptibility to infection and dilutions of a VRE phagepreparation (1.0 ml) are mixed and then added to 4.5 ml 0.7% a moltenagar in nutrient broth at 45° C. This mixture is completely poured intoa petri dish containing 25 ml of nutrient broth solidified with 2% agar.During overnight incubation at 37° C., VRE grow in the agar and form aconfluent lawn with some VRE cells being infected with phage. Thesephages replicate and lyse the initially infected cells and subsequentlyinfect and lyse neighboring bacteria. However the agar limits thephysical spread of the phage throughout the plate, resulting in smallvisibly clear areas called plaques on the plate where bacteriophage hasdestroyed VRE within the confluent lawn of VRE growth.

The number of plaques formed from a given volume of a given dilution ofbacteriophage preparation is a reflection of the titer of thebacteriophage preparation. Also since one plaque with a distinctmorphology represents one phage particle that replicated in VRE in thatarea of the bacterial lawn, the purity of a bacteriophage preparationcan be ensured by removing the material in that plaque with a pasteurpipette (a “plaque pick”) and using this material as the inoculum forfurther growth cycles of the phage. On this basis, doing further plaqueassays on preparations of phage grown from this plaque pick, one wouldexpect all plaques to have a single appearance or plaque morphologywhich is the same as the plaque picked, a further indication of purity.Therefore this technique can not only be used to test bacteriophagepotency but also bacteriophage purity.

Streak Plate Method:

Eighteen hour LB broth cultures of the different enterococci strains tobe tested arc grown at 37° C. (resulting in approximately 10⁹ CPU/ml)and a loopful of each culture is streaked across a nutrient agar platein a single line. This results in each plate having a number ofdifferent VRE streaked across it in single straight lines of growth.Single drops of phage filtrates to be tested are applied to the steaksof each VRE growth, and the plate is incubated 6 hours at 37° C., atwhich time the steaks of the different VRE strains are examined for theability of phage to form clear areas devoid of bacterial growth,indicating lysis of that particular VRE strain by that particular phage.

The VRE host range for a given phage filtrate can be ascertained bywhich VRE streaks it is capable of causing a clear area devoid of growthand which strains of VRE the phage is incapable of doing this.

Appelman Tube Turbidity Test (from Adams, M. H. 1959. Bacteriophages.Interscience Publ. New York N.Y.):

18 hour LB broth cultures of different VRE strains are prepared. 0.1 mlof phage filtrate or a dilution thereof is added to 4.5 ml of VRE brothcultures and incubated at 37° C. for 4 hours (monophages), or 4-18 hours(polyvalent phages). Phage free VRE broth cultures are used as controls.Broth cultures which are normally turbid due to bacterial growth areexamined for the ability of the phage to lyse the VRE strain asindicated by the clearing of the culture turbidity.

The host range of a given phage can be ascertained by which VRE brothcultures the phage is capable of clearing and which broth cultures itcannot induce clearing.

Example 3 A Phage Strain is Active Against over 200 VRE Isolates

A collection of 234 VRE isolates; 187 E. faecium of which 3 strains arefrom ATCC, 41 E. faecalis strains, and 6 E. gallinarium strains as wellas 6 E. faecium strains which are vancomycin sensitive were tested forsusceptibility of infection by 7 monophages isolated as described inExample 2. Susceptibility of infection was determined by the 3techniques described. The majority of VRE strains in this collectionwere isolated from patients at the University of Maryland and BaltimoreVA Medical Centers as indicated in Example 1. Such VRE isolates weredetermined to be distinct and genetically diverse by pulsed field gelelectrophoresis typing. Of the 7 monophages, VRE/E2 and VRE/E3 have arelatively narrow host range compared to other VRE phages, but are ableto infect the small proportion of VRE strains which were resistant toother phages collected. A phage cocktail containing the above 7 VREmonophages lysed 95% of the VRE strains in the collection.

Example 4 Producing Bacteriophage-containing Compositions

0.1 ml amounts of a 18-24 LB broth culture (LB broth culture containsBacto LB Broth. Miller (Luria-Bertani, dehydragted) reconstitutedaccording to instructions by Difco Laboratories, Detroit, Mich.) of astrain of VRE, which has been previously selected on the basis of beingable to produce a maximum yield of bacteriophage are mixed with 1.0 mlof a VRE monophage filtrate and then mixed with 4.5 ml of 0.7% moltenagar in nutrient broth at 45° C. This mixture is completely poured intoa petri dish containing 25 ml of nutrient broth solidified with 2% agar.After overnight incubation at 37° C., the soft top agar layer with thephage is recovered by gently scraping it off the plate, and thisrecovered layer is mixed with a small volume of broth (1 ml per plateharvested), This suspension is centrifuged at 5,000-6,000 g for 20minutes at 4° C. and the phage containing supernatant is carefullyremoved. The supernatant is filtered through a 0.45 μm filter andcentrifuged at 30,000 g for 2-3 hours at 4° C.

The phage containing pellet is suspended in 1-5 ml of phosphate bufferand is further purified by ion exchange chromatography using a Qresource ion exchange column (Pharmacia Biotech, Piscataway, N.J.) and a0-1 M NaCl gradient in the start buffer. Phage tends to be eluted fromthe column between 150-170 mM NaCl with each fraction being assessed forthe presence of phage by standard plaque assay technique. Fractionscollected and assayed arc pooled if the phage titer by the plaque assayis no greater than 3 logs lower than the phage preparation put onto thecolumn (e.g., 10¹⁰ PFU/ml is put onto the column therefore pool onlythose fractions with titers >10⁷ PFU/ml). Pooled fractions are testedfor endotoxin by the Limulus Aiiebocyte Lysate Assay (BioWhittaker Inc.,Walkersville, Md.). Pools demonstrating >50 EU/ml of endotoxin arepassed through a Affi-prep polymyxin support column (Bio-Rad Labs,Hercules, Calif.) to remove residual endotoxin.

The phage pool is buffer exchanged against 100 mM ammonium bicarbonateusing size exclusion with Sephadex G-25 chromatography (PharmaciaBiotech). 1 ml aliquots of the purified phage are freeze dried in thepresence of gelatin and stored at room temperature. The purity of thephage preparation is assessed by a combination of electron microscopy,SDS-PAGE, DNA restriction digest and analytical ultracentrifugation.

Example 5 Determination of a Protective Dose of Bacteriophage

Establishment of Sustained VRE Colonization in a Animal Model

CD-1 mice are pretreated for seven days with 0.1 mg/ml of gentamicin and0.5 mg/ml of streptomycin in drinking water to reduce their normalintestinal flora. VRE are then administered to the mice, who have fastedfor 6 hours, by consumption of one food pellet inoculated with 10⁶ CFUof VRE. VRE intestinal colonization is confirmed in mice by standardcolony counts of >10³ CFU VRE/gram of feces on CNA agar containing 10μg/ml of vancomycin, 1 μg/ml of amphotericin B and 10 μg/ml ofgentamicin. The colonization procedure is considered successful if thereis consistent shedding of >10³ CFU of VRE per gram of feces for 5-7 daysafter consumption of the spiked food pellet. VRE colonization maypersist for 4 weeks by this method. Mice are given drinking watercontaining the above mixture of antibiotics throughout the duration ofthe experiment.

Use of a to Vivo Mouse Model to Demonstrate Efficacy of LyticBacteriophage in Reducing VRE Gastrointestinal Colonization.

Twenty-four hours after detecting>10³ CFU VRE/gram of feces, mice wereadministered VRE phage (by having there consume one food pelletinoculated with 10⁹ PFU of VRE). Control groups consisted of (1)non-VRE-colonized mice sham dosed (no phage in dose), (2) VRE-colonizedmice which are sham dosed, and (3) non-VRE-colonized mice dosed withphage. Five mice were used in each group.

The efficacy of phage treatment to reduce VRE gastrointestinalcolonization was determined by quantitating VRE, on a daily basis, inweighed fecal samples from the mice in the different groups. Inaddition, at the end of the experiment, mice were sacrificed and thenumber of VRE and phage in their liver, spleen, and blood determined. Ifadministration of phage reduced VRE gastrointestinalcolonization/overall load in mice by at least 1 log as compared to thecontrol groups within 48-98 hours after phage administration, then thisdose of the particular phage was deemed efficacious. More preferably,colonization was reduced by at least 3 logs.

Example 6 Isolation and Characterization of Lytic Phages AgainstSelected Salmonella Serotypes

Isolation and purification of bacteriophages. Salmonella-specificbacteriophages were isolated, by standard techniques, from variousenvironmental sources in Maryland. Purification was performed by acombination of low- and high-speed centrifugation and by sequentialfractionation with various chromatographic media. Purified phages werebuffer-exchanged against physiological phosphate-buffered saline, pH7.6. The final product was sterilized using a 0.22 micron filter,titered, and stored in sterile glass ampules at 40C.

Bacteriophage isolates were tested against a strain collection whichconsisted of 245 Salmonella strains, including S. hadar (84 strains), S.typhimurium (42 strains), S. enteritidis (24 strains), S. heidelberg (2×strains) and S. newport (18 strains). Forty-four of the remaining 56strains were grouped in 17 serotypes and 12 strains were untypable.Genetically, this was a diverse strain population encompassing 78 PFGEtypes.

Seven clones of Salmonella-specific lytic bacteriophages were isolatedfrom environmental sources. Electron microscopy identified them as“tailed phages” of the family Myoviridae and Siphoviridae. The mostactive phage clone lysed 220 (90%) of the strains, including all DT-104(multi-drug resistant) Salmonella isolates. The second most active phagelysed 74% of the strains.

Pulsed field gel electrophoresis (PFGE). The rapid PFGE proceduredeveloped for typing E. coli 0157:H7 strains was used for PFGE typing ofthe Salmonella strains [5]. All strains were analyzed after digestingtheir DNA with Xba I, and selected strains were also analyzed afterdigesting their DNA with Avr II and Spe I restriction enzymes. TheCDC-standard S. newport strain am01144 (Xba I-digested) was used as thereference strain in all experiments. Since the number of Salmonellaestrains per PFGE type was limited, it was not determined whether therewas an association between certain clonal groups andresistance/susceptibility to these phages.

The “target range” was further increased by 5% by constructing a“cocktail of phages” consisting of three phages. This “cocktail” wasefficacious in reducing Salmonella counts on experimentally contaminatedsurfaces, and spraying 1×10⁵ PFU of phage reduced the numbers ofSalmonella from 1×10⁷ CFU to undetectable levels in less than 48 h. Thephage clones and the cocktail were not active against other bacterialspecies tested, including E. coli, P. aeruginosa, S. aureus, K.pneumoniae and L. monocytogenes, which suggests that their activity isconfined to the Salmonella species.

Environmental decontamination studies. The bottoms of approximately twoautoclaved plastic boxes (A and B) comprising approximately 225 cm² eachin surface area were evenly covered with a test Salmonella strain (1×10⁷CFU). After 1 hour, box A was sprayed with approximately 3 ml of anaqueous suspension of a Salmonella phage (1×10⁷ PFU/ml), and box B wassprayed with 3 ml of sterile water. Swab samples were taken at 3, 6, 24and 48 hours, and they were assayed, by standard techniques, todetermine the numbers of Salmonella and phage.

In the environmental decontamination studies, 3 hours after phagetreatment there was a significant reduction of approximately 2.5 logs inthe number of Salmonella on box A, as compared to the “no phage” box B.Salmonella was not detectable on the phage-exposed box (box A) after24-48 h, which corresponds to at least a 3 log drop in counts (comparedto the group that was not treated with phages). We have conductedadditional experiments examining the effect of phages on (i) variousconcentrations (1×10⁵ and 1×10³ CFU) of Salmonella, and (ii) variousconcentrations (1×10⁵ and 1×10³ CFU) of a mixed Salmonella contamination(3 strains of different serotypes). In all cases, phages reduced theSalmonella to undetectable levels in 24-48 h. Testing after prolongedexposure (10 days) indicated that there was no regrowth of Salmonella,and the phages were still detectable at low (approximately 1×10¹ PFU)levels. These data suggest that Salmonella-specific phage preparationsmay have utility in reducing/eliminating Salmonella contamination fromenvironmental surfaces, and, therefore, may be useful in decontaminatingpoultry plants, chicken houses, etc.

Finished poultry product decontamination studies: Chickens purchased atretail (2 chickens per group) were experimentally contaminated with arifampin-resistant, phage-sensitive Salmonella strain (1×10³ CFU perbard), and they were kept at room temperature for 1 hour. A phagecocktail (10 ml, 1×10⁷ PFU/ml) was sprayed on the chickens in group 3A,and the chickens in group 2A were sprayed with sterile water. Thechickens were analyzed for the presence of the test Salmonella strainusing the USDA/FSIS standard methodology for Salmonella detection.

The results of the finished poultry product decontamination studiesshowed that the number of Salmonella recovered from the phage-treatedgroup (group 3A) was approximately 10³-fold less than that recoveredfrom the; phage-untreated, control group (group 2A). These data suggestthat Salmonella-specific phages may have utility in final poultryproduct clean up; i.e., reduce/eliminate residual Salmonellacontamination of post-chill birds.

Carefully constructed, potent, Salmonella-specific phage preparationscontaining one or more lytic monophages may have utility inreducing/eliminating Salmonella contamination from environmentalsurfaces, and, therefore, may be useful in decontaminating poultryplants, chicken houses, etc. Moreover, Salmonella-specific phages may beuseful in final poultry product clean up; i.e., reduce/eliminateresidual Salmonella contamination of post-chill birds.

Example 7 Bacteriophage Sanitation of Freshly-Cut Produce

A study was performed to determine (i) the survival and growth ofSalmonella enteritidis (choleraesuis) on fresh-cut apple and honeydewmelon slices under the conditions (temperature, humidity, and length ofincubation) likely to be encountered during their processing andstorage, and (ii) the effectiveness of specific phages for use as abiocontrol agent on fresh-cut fruits contaminated with Salmonella.

Fruit. All of the fruits were disinfected with 70% EtOH before slicing.“Red Delicious” apples stored at 1° C. were cut into eight slices withan apple slicer and wounded (Conway, W. S., B. Leverentz, R. A. Saftner,W. J. Janisiewicz, C. E. Sams, and E. Leblanc “Survival and growth ofListeria monocytogenes on fresh-cut apple slices and its interactionwith Glomerella cingulata and Penicillium expansum” Plant Disease84:177-181 (2000)). Honeydew melons purchased from a local supermarketwere sliced through the equator with a sterile knife. Two rings were cutout of the center of each melon, and each ring was cut into 12 equalslices. The pH ranges of the apples and honeydew melon tissuesdetermined with a pH combination electrode, Semi-Micro (81-03 ROSS™,Orion Research, Inc., Beverly, Mass.). were pH 4.1-4.7 and pH5.7-5.9,respectively.

Preparation of the bacterial inoculum. A rifampicin-resistant, phagepreparation-susceptible Salmonella enteritidis strain, from thebacterial strain collection of Intralytix, Inc. (Baltimore, Md.), wasused to experimentally contaminate the apple and honeydew melon slices.The bacterium was grown overnight at 37° C. on L-Agar supplemented with100 -μg/ml rifampicin (Sigma #R-3501), the bacteria were collected andwashed with sterile saline (0.9% NaCl), and the bacterial suspension wasdiluted to a concentration of 1×10⁶ CFU/ml.

Phage. The phage mixture (SCPLX-phage). containing 4 distinct lyticphages specific for Salmonella enteritidis was obtained from Intralytixat a concentration of 10¹⁰ PFU/ml in phosphate-buffered saline. Themixture was diluted with sterile saline (10⁷ PFU/ml finalconcentration), immediately before applying onto the fruit slices.

Bacterial inoculation and phase application. Twenty-five μl of thebacterial suspension were applied to wounds made in the fruit slices.After applying the Salmonella strain, 25 μl of the phage mixture wereapplied to the wounds, and the slices were placed in 475-ml Mason jarscovered with plastic film. Real View laboratory sealing film (NortonPerformance Plastics, location?) was used to seal jars containing theapple slices and a Std-Gauge film with a high oxygen transfer rate typeLDX5406, product 9NK27 (Cryovac, Duncan, S.C.) was used to seal the jarscontaining the honeydew melon slices.

Recovery of bacteria and phages. After inoculation, the Mason jarscontaining the fruit slices were stored at 5, 10 and 20° C. The numberof CFU/ml on the apple and honeydew melon slices was determined at 0, 3,24, 48, 120, and 168 h (4 fruit slices per treatment for each recoverytime) after inoculation. Recovery and quantitation of the bacteria wasperformed according to the procedure described previously. After platingthe samples, the remaining sample solution was filter-sterilized (0.45μm Supor membrane, Acrodisk, Pall Gelman) and stored at 4° C. The titerof the phage in this filtrate was determined according to standardprocedures (Adams, M. H. “Bactenrophages” Interscience Publishers, NewYork. (1959)). All experiments were repeated at least twice to ensurereproducibility.

RAPD and PFGE. The RAPD technique was performed, according to themanufacturer's instructions, using a RAPD kit (Amersham PharmaciaBiotech, Piscataway, N.J.) containing ready-to-go analysis beads, andthe DNA patterns were analyzed by electrophoresis in 2% agarose gel inTAE buffer. PFGE was performed using the CHEF Mapper (Bio-RadLaboratories, Hercules, Calif.), as described previously .

Statistical analyses. The numbers of CFU/wound on apple slices wereanalyzed as a three-factor general linear model using PROC MIXED(SAS/STAT® Software: Changes and Enhancements through Release 6.12, pp.1167. Cary, N.C. 1997 (“SAS Institute”)) with treatment, temperature andtime as the factors. The assumptions of the general linear model weretested. To correct variance heterogeneity, the values were log₁₀transformed, (log x) and treatments were grouped into similar variancegroups for the analysis. The means were compared using pair-wisecomparisons with Sidak adjusted p-values so that the experiment-wiseerror for the comparison category was 0.05.

The analysis for the honeydew data was done in two parts, since thevalues for 5° C. at 120 and 168 h were all zero.

Part 1: The)CFU values for 0, 3, 24, and 48 h were analyzed as athree-factor general linear model using PROC MIXED (SAS Institute) withtreatment, temperature and time as the factors. The assumptions of thegeneral linear model were tested. To correct variance heterogeneity, thevalues were log₁₀ plus one transformed, (log (x+1)) and treatments weregrouped into similar variance groups for the analysis. The means werecompared using pair-wise comparisons with Sidak adjusted p-values sothat the experiment-wise error for the comparison category was 0.05. Totest for the influence of time or temperature on the phage treatment,the magnitude of the difference between the phage treatment and thecontrol at each temperature at a given time was tested against thedifference for the other temperatures at the same time.

Part 2: The CFU values for 0, 3, 24, 48, 120 and 168 at 10° C. and 20°C. were analyzed as a four-factor general linear mixed model using PROCMIXED (SAS Institute) with treatment, temperature and time as the fixedfactors and experiment as the random factor. The assumptions of thegeneral linear model were tested. To correct variance heterogeneity, thevalues were log₁₀ plus one transformed, (log (x+1)) and treatments weregrouped into similar variance groups for the analysis. The means werecompared using pair-wise comparisons with Sidak adjusted p-values sothat the experiment-wise error for the comparison category was 0.05. Totest for the influence of time or temperature on the phage treatment,the magnitude of the difference between the phage treatment and thecontrol at 10° C. was tested against the difference for 20° C. at eachtime period.

Results.

a. Salmonella growth on fruit. Salmonella enteritidis survived at 5° C.and grew at 10 and 20° C. on “Red Delicious” apple slices (pH 4.1-4.7)and honeydew melon slices (pH 5.7-5.9) stored during a time of 168 h. Asexpected, the most vigorous bacterial growth was observed on thefresh-cut fruits stored at 20° C., with the number of bacteria rapidlyincreasing (by approximately 3.5 logs) on both honeydew melons and “RedDelicious” apples within the first 24 h after inoculation, and furtherincreasing on honeydew melons by additional 2 logs. In general,Salmonella grew better on honeydew melons than apples, with the mostprofound difference (approximately 2 logs) observed at 168 h between thegroups incubated at 20° C. At a lower temperature (4° C.), cellpopulations were stagnant and the Salmonella did not grow noticeably oneither of the fresh-cut fruits tested; on honeydew melons, the bacterialpopulation actually decreased starting from 120 h of incubation.

Several steps were taken to ensure that no wild-type Salmonella strains(that initially may have been present on the fruit surface) werecultured. For example: (i) the fruits' uncut surfaces were cleaned with70% ethanol at the beginning of each experiment, and (ii) rifampin (150μg/ml) was included in the selective media, in order to ensure that onlythe original, rifampin-resistant test strain was quantitated. Inaddition, 10-15 randomly selected colonies were analyzed by RAPD and/orPFGE after each experiment, and the patterns were compared to that ofthe test S. enteritidis strain.

b. Phage persistence on fruit. The mixture of Salmonellaenteritidis-specific phages continually declined by about 3 log units onhoneydew melon over a period of 168 h. This decline was similar for alltemperatures. In contrast, the phage concentration on the apple slicesdecreased by approximately 6 log after 3 h, the phage could not bedetected after 24 h at 10 and 20° C. and after 48 h at 5° C. In order todetermine whether different acidity of “Red Delicious” apples (pH 4.2)and honeydew melons (pH 5.8) was responsible for this difference, wedetermined phage titers in the aliquots of the SCPLX preparationincubated (4° C.) at pH 4.2 and 5.8 for 48 h. Approximately 4 times morephages were recovered from the aliquots incubated at pH 5.8 than fromthose incubated at pH 4.2 (data not shown).

c. Pathogen control by the phase treatment. The bacterial count wasconsistently lower (by approximately 3.5 logs) on the honeydew melontreated with the phage mixture than on corresponding samples of thecontrol. There was no significant difference between the numbers ofSalmonella on the apple slices in the control and test groups. Ingeneral, the effect of the phage mixture was independent of temperatureand time during the duration of the experiment (see Table 1, below). Theonly significant effect attributed to temperature occurred at 48 h ofincubation, when the phage mixture suppressed S. enteritidis populationson honeydew melon more at 10° C. than at 20° C. (see Table 2, below).Statistical analysis of the differences between the treatments atvarious times and temperatures did not reveal any other effect of theseparameters on the phage treatment of honeydew melon (see Table 3,below). Phage susceptibility testing of the bacteria that survived phagetreatment indicated that they did not develop resistance against phagesin the SCPLX preparation. TABLE 1 Log (CFU) Mean Comparisons forHoneydew honeydew treatment part 1 part 2 control 3.17a* 4.97a* phagetreatment 1.38b 3.74b*Treatment means with different letters are different at significancelevel ≦ 0.0001.

TABLE 2 Comparisons of Treatment Differences between Temperatures at aSpecific Time on Honeydew p-value time [h] 5 vs. 10° C. 5 vs. 20° C. 10vs. 20° C. part 1 0 0.2764 0.5645 0.5562 3 0.4685 0.8058 0.5473 240.1873 0.4964 0.2921 48 0.3450 0.0437 0.0039 part 2 120 n/d n/d 0.9497168 n/d n/d 0.4119

TABLE 3 Analysis of Variance p-values source ‘Red Delicious’ honeydewpart 1 honeydew part 2 treatment 0.0060 0.0001 0.0001 temperature 0.00010.0001 0.0001 trt × temp 0.0001 0.3594 0.3594 time 0.0001 0.0001 0.0001trt × time 0.0060 0.2388 0.2388 temp × time 0.0001 0.0001 0.0001 trt ×temp × time 0.0818 0.2556 0.2556Deposit Information

Six bacteriophages have been deposited under the Budapest Treaty. Thesedeposits were made with the American Type Culture Collection (ATCC),10801 University Boulevard, Manassas, Va. 20110. These bacteriophagesare identified, as follows: Phage SA-36 SPT-1 MSP-71 Phage LIST-3 ENT-7ECO-9

For purposes of clarity of understanding, the foregoing invention hasbeen described in some detail by way of illustration and example inconjunction with specific embodiments, although other aspects,advantages and modifications will be apparent to those skilled in theart to which the invention pertains. The foregoing description andexamples are intended to illustrate, but not limit the scope of theinvention. Modifications of the above-described modes for carrying outthe invention that are apparent to persons of skill in medicine,bacteriology, infectious diseases, pharmacology, and/or related fieldsare intended to be within the scope of the invention, which is limitedonly by the appended claims.

All publications and patent applications mentioned in this specificationare indicative of the level of skill of those skilled in the art towhich this invention pertains. All publications and patent applicationsare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

1. A method for sanitation of produce, comprising: providing at leastone bacteriophage effective against Vancomycin Resistant Enterococci(VRE), Staphylococcus, Pseudomonas, Campylobacter, E. coli, Salmonella,or a combination thereof; and applying the bacteriophage to the produce;wherein the produce is selected from the group consisting of: fruits,vegetables, and combinations thereof.
 2. The method of claim 1, whereinthe produce comprises at least one of freshly cut produce, damagedproduce, diseased produce, or contaminated produce.
 3. The method ofclaim 1, wherein the application of the bacteriophage to the producecomprises spraying the bacteriophage on the produce.
 4. The method ofclaim 1, wherein the application of the bacteriophage to the producecomprises misting the bacteriophage on the produce.
 5. The method ofclaim 1, wherein the application of the bacteriophage to the producecomprises washing the produce in the bacteriophage.
 6. The method ofclaim 1, wherein the application of the bacteriophage to the producecomprises immersing the produce in a solution containing thebacteriophage.
 7. The method of claim 1, wherein the application of thebacteriophage to the produce comprises periodically applying thebacteriophage to the produce.
 8. The method of claim 1, wherein theapplication of the bacteriophage to the produce comprises continuouslyapplying the bacteriophage to the produce.
 9. The method of claim 1,further comprising applying at least one chemical sanitizer to theproduce.
 10. The method of claim 9, wherein the application of thebacteriophage to the produce and the application of the chemicalsanitizer to the produce are performed substantially simultaneously. 11.The method of claim 1, wherein the application of the bacteriophage tothe produce comprises applying the bacteriophage to the produce in anamount effective to reduce colonization of pathogenic bacteriasusceptible to the bacteriophage by at least one log.
 12. The method ofclaim 1, wherein the bacteriophage comprises a bacteriophage cocktail.13. The method of claim 12, wherein the bacteriophage is targetedagainst at least one food-borne pathogen.
 14. The method of claim 13,wherein the food-borne pathogen comprises at least one of Salmonella orshiga toxin-producing E. coli.
 15. The method of claim 1, wherein thebacteriophage comprises at least one pH-resistant bacteriophage.
 16. Themethod of claim 1, further comprising applying bacteriocin nisin to theproduce.
 17. The method of claim 1, wherein the VRE is selected from thegroup consisting of: E. faecium, E. faecalis, E. gallinarium, and acombination thereof.
 18. The method of claim 17, wherein thebacteriophage is applied in an amount sufficient to result in a 1 to 3log reduction of the VRE.
 19. The method of claim 1, wherein thePseudomonas is Pseudomonas aeruginosa.
 20. The method of claim 1,wherein the Salmonella is Salmonella enteritidis.
 21. The method ofclaim 1, wherein the Staphylococcus is Staphylococcus aureus,Staphylococcus epidermidis, or a combination thereof.